The overground parts of A. glycyphyllos were harvested in July 2021 from Vitosha Mt., Bulgaria. Prof. D. Pavlova (Department of Botany, Faculty of Biology, Sofia University) identified the plant, and a voucher specimen (SO-107613) is kept in the Herbarium of the same university for further reference. The air-dried plant material (200 g) was powdered (3 mm) and then extracted with dichloromethane (6 × 2 L) using percolation to remove the lipophilic constituents. The defatted plant substance was then aired and exhaustively extracted with 80% MeOH (24 × 3 L) using percolation. The obtained extract was filtered, concentrated under vacuum, and then lyophilized to produce a dry extract (42 g). The extract was separated over a Diaion HP-20 (4.7 × 45 cm) column, eluting with H₂O: MeOH (0→100%). Seven main fractions were collected (I–VII). After the TLC analysis (Silica gel plates, EtOAC:HCOOH:AcOH:H₂O = 32:3:2:6, anisaldehyde/ c. H₂SO₄, 104 °C, 10 min), fraction VI was found to be rich in saponins. It was chromatographed on a silica gel cartridge using flash chromatography with CH₂Cl₂:MeOH:H₂O (step gradient 8:2:0.2→7:3:0.3) to obtain 21 subfractions. Subfraction 12, which contained a main compound (TLC), was further separated with CH₂Cl₂:MeOH:H₂O (step gradient 9:1:0→5:6:1) using flash chromatography on a silica gel cartridge and a compound, named Sg, was obtained.

A Q Exactive Plus Orbitrap mass spectrometer with a heated electrospray ionisation (HESI) ion source (ThermoFisher Scientific, Bremen, Germany) coupled with a UHPLC system (Dionex UltiMate 3000 RSLC, ThermoFisher Scientific, Bremen, Germany) was used. The full scan MS was set at: resolution 70 000 (at m/z 200), AGC target 3e⁶, max IT 100 ms, scan range 250 to 1700 m/z. The MS² conditions were: resolution 17 500 (at m/z 200), AGC target 1e⁵, max IT 50 ms, mass range m/z 250 to 1750, isolation window 2.0 m/z and (N)CE 20. The ionization device (HESI source) was operating at +3.5 or -2.5 kV spray voltage and 320 °C capillary and probe temperature, 38 arbitrary units (a.u., as set by the Extactive Tune software) of sheath gas and 12 a.u. of auxiliary gas (both N₂); S-Lens RF level 50.0. UHPLC separations were performed on a Kromasil C18 column (1.9 μm, 2.1 × 50 mm, Akzo Nobel, Sweden) at 40 °C. The mobile phase was (A) H₂O + 0.1% HCOOH and (B) MeCN + 0.1% HCOOH, and the flow rate was 0.3 mL/min. The elution was as follows: 10% B for 1.5 min, increase to 30% B for 1 min, isocratic with 30% B for 0.5 min, increase to 95% B for 9 min, isocratic with 95% B for 2 min, return to 10% B for 0.1 min. Identification was supported by MS² experiments, which revealed the aglycone part of the molecule as well as the successive loss of monosaccharides of the sugar moiety. The fragmentation pattern was compared to literature data. The software Xcalibur, Version 4.2 (Thermo Scientific) was used for data collection and processing.

Monoamine oxidase activity assay of both recombinant human MAOA (hMAOA) and MAOB (hMAOB) was performed using a fluorimetric method with small modifications (Held and Buehrer 2003; Dezsi and Vecsei 2017; Kasabova-Angelova et al. 2020) by Amplex UltraRed reagent (Yagi 1976) to detect hydrogen peroxide (or peroxidase activity) in biological samples (or enzymes). In the presence of peroxidase, Amplex Red reacted 1:1 stoichiometrically with hydrogen peroxide to produce a red phosphorescent oxidation product (resorufin). Due to the high extinction coefficient (58,000 ± 5,000 cm⁻¹.M⁻¹), the analysis could be performed fluorometrically or spectrophotometrically. The reaction was used to detect small amounts of hydrogen peroxide (on the order of 10 pM, in a volume of 100 µL). Pure MAOA or MAOB working solution in reaction buffer, MAOA or MAOB working solution containing hydrogen peroxide, and pure reaction buffer were used as controls.

The substances (the saponin Sg at 0.050, 0.250, 0.500, 075 and 1 µM, or Chlorgyline or Selegiline, 1 µM), together with hMAOA or hMAOB, were put in a 96-well plate (8 samples for each substance), after which the plate was placed in an incubator for 30 min (in the dark, at 37 °C). At the end of the incubation period, the reaction was initiated by adding to each well on the 96-well plate a 50 µL a Mix Solution containing solutions of Amplex Red reagent, Horseradish Peroxidase (HRP) and tyramine as substrates for the enzymes, in a reaction buffer. As the reaction was continuous, fluorescence was monitored every 30 min (0, 30, 60, 90, 120 and 150 min, respectively) to scrutinize kinetics in the dark as the reaction mixture was shaken at a constant temperature of 37 °C. Fluorimetric reading was performed on a Synergy 2 Microplate Reader at two different wavelengths (λ = 570 and 690 nm).

The enzyme activity was expressed as a normalized percent of the untreated control set as 100% and the results were expressed as mean values and standard deviations (± SD) (Graph Pad Prizm, v. 6). Statistical analysis was performed by one-way analysis of variance (ANOVA) with the post hoc multiple comparisons procedure (Dunnet’s test) to assess the statistical differences in the normal distribution. Values of P < 0.05, P < 0.01 and P < 0.001 were considered statistically significant.

From a defatted extract of the aerial parts of A. glycyphyllos, using repetitive column and flash chromatography over different sorbents, one compound (Sg) was isolated as a white powder (3 mg) with a purity of 98.2% (UHPLC-MS) (Fig. 1).

Figure 1. 

Chromatograms of Sg in the negative (a) and positive (b) ionization modes.

From its chromatographic properties, the coloration in TLC analysis, the MS data, it was proven that Sg is a triterpenoid saponin. The structural elucidation was performed by means of HRESIMS and an analysis of the fragmentation pattern. In the negative mode, a deprotonated molecular ion was observed [M-H]- m/z 941.5115, C₄₈H₇₇O₁₈-, and in the positive, a protonated one ([M+H]⁺ m/z 943.5261, C₄₈H₇₉O₁₈⁺), which is consistent with a molecular formula of C₄₈H₇₈O₁₈ (942.5188) (Fig. 2). When these ions were subjected to MS² analysis, a successive loss of monosaccharides was noted. Firstly, a methylpentose (Stobiecki 2000) unit (m/z 146.0579, C₆H₁₀O₄) was cleft: [M-H-(mepent)]-, m/z 795.4536, C₄₂H₆₇O₁₄-, and [M+H-(mepent)]⁺, m/z 797.4682, C₄₂H₆₉O₁₄⁺. The second loss was of a hexose (Stobiecki 2000) unit (m/z 162.0528, C₆H₁₀O₅), resulting in the following ions: [M-H-(mepent+hex)]-, m/z 633.4008, C₃₆H₅₇O₉-, and [M+H-(mepent+hex)]⁺, m/z 635.4154, C₃₆H₅₉O₉⁺. At the third cleavage, a hexuronic acid (Hvattum and Ekeberg 2003) moiety (m/z 176.0321, C₆H₈O₆) was lost: [M-H-(mepent+hex+hexUA)]-, m/z 457.3687, C₃₀H₄₉O₃-, and [M+H-(mepent+hex+hexUA)]⁺, m/z 459.3833, C₃₀H₅₁O₃⁺ (Fig. 2).

Figure 2. 

HRESIMS spectrum of compound Sg in the (a) negative and (b) positive modes.

The final fragment was the sapogenin of compound Sg, and the values were consistent with soyasapogenol B (C₃₀H₅₀O₃, 458.3759) (Zhang and Popovich 2010). From the MS analysis, it can be concluded that Sg is a trisaccharide of soyasapogenol B, with a sugar chain/s consisting of a methylpentose, a hexose, and a hexuronic acid. Further spectral analysis of the isolated compound is necessary to determine the place and order of attachment of the sugars. Nevertheless, the aim here was to explore its pharmacological potential.

On the activity of the hMAOA enzyme, Sg did not reveal a statistically significant inhibitory effect. In this study, only the classical MAOA inhibitor, Chlorogyline, decreased statistically significant enzyme activity as follows: 0.050 μМ – by 15%; 0.250 μМ – by 30%; 0.500 μМ – by 35%; 0.750 μМ – by 45%; and 1 μМ – by 50%, compared to the control (pure, non-treated hMAOA) (Fig. 3).

Figure 3. 

Effect of Sg and Chlorgyline (at concentrations of 0.050, 0.250, 0.500, 0.750, and 1 μM) on the activity (%) of human recombinant MAOA enzyme (hMAOA). * P < 0.05; ** P < 0.01; *** P < 0.001 vs. control (pure hMAOA).

On hMAOB activity, Sg exhibited a statistically significant inhibitory effect only at concentrations of 0.750 μM and 1 μM. At a concentration of 0.750 μM, the saponin inhibited the enzyme by 25%, and at 1 μM by 35%, compared to the control (pure hMAOB). The effects of Sg at these concentrations were close to those of Selegiline. This classical MAOB inhibitor statistically significantly reduced the enzyme activity as follows: 0.050 μM by 20%; 0.250 μM by 35%; 0.500 μM by 40%; 0.750 μM by 50%; and 1 μM by 55% compared to the control (pure hMAOB) (Fig. 4).

Figure 4. 

Effect of Sg and Selegiline (at concentrations of 0.050, 0.250, 0.500, 0.750, and 1 μM) on human recombinant MAOA enzyme (hMAOB) activity. ** P < 0.01; *** P < 0.001 vs. control (pure hMAOB).

MAO-induced oxidative stress is a potential risk factor for neuronal loss in aging and is associated with age-related neurodegenerative disorders, including Parkinson’s disease (PD). It is known that mitochondrial DNA is damaged by increased levels of oxidative products due to increased MAO activity (Hauptmann et al. 1996). In a mouse model, the leading pathogenetic cause of PD is reported to be the upregulation of MAOB in astrocytes (Mallajosyula et al. 2008, 2012). Inhibition of MAO increases the content of amines in the brain, resulting in antidepressant effects and improved neuronal activity (Youdim et al. 2006; Fišar 2016). In this study, the isolated saponin exhibited a concentration-dependent inhibiting effect on hMAOB activity.

From a pharmacological point of view, the identification of novel possible compounds acting as selective MAO inhibitors is of great importance. Thus, the research interest worldwide is high. It is known that the selective MAOB inhibitors Selegiline and Rasagiline have shown a protective action on neuronal cells in both in vitro and in vivo models (Ebadi et al. 2006; Youdim et al. 2006; Makoto Naoi and Inaba-Hasegawa 2013). These drugs have been approved for many years as adjunctive therapy in PD, preserving dopamine levels in the brain and thus postponing the need for L-DOPA. Another function of MAO is the deamination of GABA, which is proven in very high concentrations in vitro (Goldberg and Pollard 2014).

The results of the current study are consistent with the literature. There are other reports on the hMAOB inhibiting properties of cycloartane-type triterpenoid saponins from A. glycyphyllos (Stambolov et al. 2023a, b; Shkondrov et al. 2020). Unlike these, this is the first instance of an oleanane-type saponin, isolated from Astragalus species, exhibiting these effects.

Our findings support the importance of plant-derived secondary metabolites such as triterpene saponins as lead molecules for the possible treatment of neurodegenerative diseases. Further analysis is needed to deepen our knowledge of these valuable molecules.