Antiproliferative activity of extract from in vitro callus cultures of Astragalus vesicarius ssp. carniolicus (A. Kern.) Chater

¬ Five isoflavonoids, i.e. 5-hydroxy-7-methoxy-2’, 5’-dihydroxyisoflavone (AV4), 5, 7-dihydroxy-4’-methoxyisoflavone (AV6), 7-methoxy-5-hydroxy-4’-methoxy-2’-hydroxyisoflavone (AV7), 8-pregnyl genistein (AV9), 5,7-dihydroxy-8-pregnyl-4’-methoxy-2’-hydroxyisoflavone (AV10) and one coumarochromone – sophorophenolone (AV8) were isolated from EtOAc of in vitro callus cultures of Astragalus vesicarius ssp. carniolicus, after enzymatic hydrolysis with β-glucosidase. Their structures were tentatively elucidated by spectroscopic mean (1H NMR and HR-ESI-MS spectra). Antiproliferative activity of EtOAc extract and isolated aglycones against chemosensitive human promyelocyte cell line HL-60 and its multidrug-resistant variant HL-60/Dox was assessed in vitro. Despite the strong activity of EtOAc (IC50 8.8 μg/mL (HL-6, 72 h) to 11.8 μg/mL (HL-60/Dox, 72 h)), prenylated compound AV9 showed also antiproliferative activity – 36.1 μg/mL (HL-60 and HL-60/Dox, 72 h).


Introduction
Astragalus L. Fabaceae (Leguminosae) is the largest genus among angiosperms. It consist of about 3000 species distributed on all continents except Australia. The center of origin and biodiversity of plants of the genus Astragalus is Eurasia, and in particular, the mountainous parts of Southwest Asia. It is found mainly in Southwest and Central Asia (1500 species), the Sino-Himalayan region, North America (500 species), the Andes in South America (150 species) and Europe (133 species) (Podlech 2008;Benchadi et al. 2013). The Bulgarian flora is represented by 31 species, including local endemics, nine of which are included in the Red Book of Bulgaria. Endemic plants are an emblematic symbol of the Bulgarian flora and one of the most sensitive and vulnerable units in the natural ecosystems of the country (Anchev 2011). Recently, the interest in the chemical composition of species of the genus Astragalus has increased significantly and various flavonoids, amino acids, saponins, alkaloids, polysaccharides, sterols has been established (Xiaoxia et al. 2014). 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 Astragalus hamosus (Krasteva et al. 2008). Flavonoids from the seeds of Astragalus complanatus inhibited the growth of human hepatocarcinoma (SMMC-7721 and HepG2) cells in vitro (Hu et al. 2009).
In this context, as alternative methods for production of secondary metabolites appear plant cell and tissue culture techniques. 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 from 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 these advantages, there are metabolites that can be produced by in vitro cultures but generally not found in the intact plants (Pavlov et al. 2005).
Thus, the focus of the present study is to determine the antiproliferative activity of EtOAc extract and isolated aglycones from in vitro callus cultures of A. vesicarius ssp. carniolicus.

General experimental procedures
Solvents were obtained from Fischer Chemicals (Loughborough, UK) and were at least of analytical grade, whereas solvents used for semi-preparative HPLC analysis, i.e. ACN and MeOH were HPLC grade and were purchased from Fischer Scientific (Loughborough, UK). Fischer Scientific (Loughborough, UK) supplied formic acid (FA). Deuterated solvents as MeOD (99.8 atom % D) were purchased from Sigma-Aldrich (Germany). H 2 O was prepared by a Milli-Q system, Millipore (Bedford, MA, USA) and filtered through 0.22 μm membrane filter. Series of chromatographic separation were performed with Diaion HP-20 (Supelco, USA) and Silica gel 60-200 µm (Merck -Millipore, Germany) column chromatography (CC). HPLC system Young Lin 9100 (Hogye -dong, Anyang, Korea) consisting of vacuum degasser, YL9110 quaternary pump, YL 9160 PDA photodiode detector, hand injector and YL Clarity software was used to perform semi-preparative column chromatography and isolation of pure compounds. 1 H NMR spectra were recorded on a Bruker AVII+ 600 spectrometer (Bruker, Karlsruhe, Germany), operating at a proton NMR frequency of 600.13 MHz. LC-MS analysis of pure compounds were performed at Thermo Scientific Q Exactive plusquadrupole -Orbitrap mass spectrometer used in ultra-high resolution mode (70 000, at m/z 200) coupled with a UPLC Dionex Ultimate 3 000 RSLC system equipped with a RP-18 Kinetex column (2.10 mm × 100 mm, 2.6 µm, Phenomenex (Corporation, Torrence, CA, USA). MS grade solvents ACN and H 2 O were used (Fischer). Gradient elution (1.4 min 10% ACN; 7 min 40% ACN; 10 min 100% ACN) of filtered and degassed ACN/H 2 O solution of FA 0.1% (v/v); column temperature 30 °C; a flow rate of about 300 µl/min was used during the analysis. The operating conditions of the HR-ESI source ionization device were: 3.5 kV voltage and 320 °C capillary temperature, 25 units of carrier gas flow and 5 units of dry gas flow. All other detector parameters were set in such a way as to obtain the most intense signal from [M+H] + . Nitrogen was used to atomize the samples. All data were recorded and processed using Xcalibur software, version 2.0 (Thermo Fisher).

Plant material
Callus cultures from A. vesicarius ssp. carniolicus were successfully established and maintained in our lab (Zarev et al. 2019).

Biological evaluation
The cell survival of all six pure compounds and EtOAc extract were tested against chemosensitive human promyelocyte cell line HL-60 and its multidrug-resistant variant HL-60/Dox. Concentration ranged from 0.001 µg/mL to 100 µg/mL.

Identification of isolated compounds
Based on the 1 H NMR data (Table 1), HR-ESI-MS spectra and UV spectrum (Suppl. material 1: Fig. S1) tentatively were identified five derivatives of isoflavones and one coumarochromone. Compound AV4 was isolated as a white powder (0.5 mg) and a protonated molecular ion with m/z 301.0700 [M+H] + was observed in HR-ESI-MS analysis, corresponding to a molecular formula of C 16 H 13 O 6 (calcd for m/z 301.0707) (Suppl. material 1: Fig. S2). The UV spectrum of the compound AV4 shows two maxima at 260 nm and 290 nm (Suppl. material 1: Fig. S1). Observing a single proton δ H 8.10 (s, 1H, H-2) and UV maximum of 260 nm, we assume the structure to isoflavonoids (Fig.  1). The presence of two protons with the following chemical shifts and coupling constants δ H 6.60 (d, J 2.29 Hz, 1H, H-5) and δ H 6.43 (d, J 2.36 Hz, 1H, H-7) is associated with C-5 and C-7 substitution in ring B of compound AV4. A singlet at δ H 3.92 (s, 3H) is characteristic for methoxy group in the molecule. Considering the coupling constants of the remaining protons in the aromatic region of the spectrum, it is visible the presence of two vicinal protons at δ H 7.09, (d, J 8.20 Hz, 1H, H-3') and 6.40, (dd,J 2.43,8.51 Hz,1H,. The proton at H-4' is a doublet of doublets with a low value of the spin-spin constant, which is marker for H-atom at meta position δ H 6.41 (d, 2.07, 1H, H-6') (Moco et al. 2006). Therefore, the structure of compound AV4 was established as 5-hydroxy-7-methoxy-2' , 5'-dihydroxyisoflavone (Suppl. material 1: Fig. S3 and Suppl. material 1: Fig. S4).
Compound AV6 was isolated as a white powder (0.1 mg) and a protonated molecular ion with m/z 285.0752 [M+H] + was observed in HR-ESI-MS analysis, which corresponded to a molecular formula C 17 H 14 O 6 (calcd for m/z 285.0758) (Suppl. material 1: Fig. S5). The UV spectrum of the compound showed three maxima at 202 nm, 262 nm and 294 nm (Suppl. material 1: Fig. S1). Similarly, to compound AV4 in the 1 H NMR spectrum of compound AV6, a single proton at δ H 8.12 (s, 1H, H-2) proved the isoflavonoid skeleton (Fig. 1). The arrangement of the substituents in ring A is also identical to that of compound AV4, which is confirmed by the presence of two protons with low values of the spin-spin constant located in the aromatic region -δ H 6.59 (d, J 2.38, 1H, H-5) and δ H 6.40 (d, J 2.20, 1H, H-7) (Suppl. material 1: Fig. S6 and Suppl. material 1: Fig. S7) 1: Fig. S8). The UV spectrum of the compound showed two maxima at 262 nm and 294 nm (Suppl. material 1: Fig. S1). The chemical shifts in the 1 H NMR spectrum are similar to those for compound AV4, as well as coupling constants (Table 1). The difference between both compounds is the presence of an additional methoxy group in AV7 δ H 3.93 (s, 3H). Thus, compound AV7 is defined as 7-methoxy-5-hydroxy-4'-methoxy-2'-hydroxyisoflavone (Suppl. material 1: Fig. S9 and Suppl. material 1: Fig. S10). Compound AV8 was isolated as a white powder (0.4 mg) and a protonated molecular ion with m/z 299.0545 [M+H] + was observed in HR-ESI-MS analysis, which corresponds to a molecular formula C 16 H 11 O 6 (calcd for m/z 299.0550) (Suppl. material 1: Fig. S11). The UV spectrum of the compound showed three maxima at 256 nm, 282 nm and 338 nm (Suppl. material 1: Fig. S1). In the MS/MS spectra of compound AV8 was observed increase of a RDB value with one unit, suggesting the presence of one more heterocycle in the structure when compared to the compounds described above. The lack of a characteristic singlet for a proton atom at H-2 or H-3 position is another evidence of the formation of an additional heterocycle in the molecule. Like compound AV4, the location of the substituents in ring A is determined on the basis of the chemical shifts and coupling constants -δ H 6.76 (d, J 2.24, 1H, H-6), δ H 6.50 (d, J 2.25, 1H, H-8). Protons from the aromatic region and their spin-spin constants are characteristic of substitution in 4' position δ H 7.07 (d, J 2.00, 1H, H-3'), δ H 6.96 (dd J 2.10, 8.40, 1H, H-5'), δ H 7.85 (d, J 8.34, 1H, H-6'). Signal at δ H 3.94 (s, 3H) also indicates the presence of a methoxy group in the molecule, thus the compound AV8 is defined as sophorophenolone (Suppl. material 1: Fig. S12 and Suppl. material 1: Fig. S13) (Tang et al. 2002).
Substance AV10 was isolated as a white powder (0.4 mg) and a protonated molecular ion with m/z 369.1329 [M+H] + was observed in HR-ESI-MS analysis, which corresponds to a molecular formula C 21 H 21 O 6 (calcd for m/z 369.1338) (Suppl. material 1: Fig. S16). The UV spectrum of the compound shows two maxima at 267 nm and 347 nm (Suppl. material 1: Fig. S1). Similarly to the above described compound, in the 1 H NMR spectrum of compound AV10, a single proton at δ H 8.22 (s, 1H, H-2) was observed as well as signals for prenylation at C-8. The chemical shifts of the protons from the aromatic region showed a substitution in ring B identical to that in AV7. Thus, compound AV10 was defined as 5, 7-dihydroxy-8-pregnyl-4'-methoxy-2'-hydroxyisoflavone (Suppl. material 1: Fig. S17).

Conclusion
The results herein provide additional phytochemical and biological data concerning the flavonoids isolated from Astragalus vesicarius ssp. carniolicus. Five isoflavonoids and one coumarochromone aglycones were characterized. Their antiproliferative activity was evaluated against chemosensitive human promyelocyte cell line HL-60 and its multidrug-resistant variant HL-60/Dox in MTT test. Some of those aglycones as well as the output for isolation EtOAc fraction displayed strong antiproliferative activity, especially prenylated derivatives such as compound AV9. Therefore, in vitro cultures of plant cells represent a promising future for the production of many valuable compounds.