Triterpenoid saponins are an important group of secondary metabolites, isolated from species of the genus Astragalus, and known for their structural variety and high polarity. They are classified into two large groups: tetracyclic and pentacyclic. It is known that most Astragalus species found in Bulgaria contain predominately oleanane-type ones (Krasteva et al. 2016). The earliest report on the plant is on sapogenins, identified after acid hydrolysis of the aerial parts. The pentacyclic sapogenins soyasapogenol B and 3β,22β,24-trihidroxyolean-12-en-19-one were reported (Elenga et al. 1986; Elenga et al. 1987). Noteworthy, to date, no corresponding saponins of these two oleananes have been isolated. Unlike these, A. glycyphyllos accumulates mainly tetracyclic cycloartane triterpenoid saponins (Table 1). From the roots of the plant, askendoside C and F were isolated (Linnek et al. 2008; Linnek et al. 2009). Recently, from the aerial parts 17(R),20(R)-3β,6α,16β-trihydroxycycloartanyl-23-carboxilic acid 16-lactone 3-O-β-D-glucopyranoside was obtained (Shkondrov et al. 2020a).

Table 1.

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Sapogenins and saponins isolated from A. glycyphyllos.

Cycloartane-type saponins
20(R),24(R)-3β,6α,16β,25-pentahydroxycycloartane			17(R),20(R)-3β,6α,16β-trihydroxycycloartanyl-23-carboxylic acid 16-lactone 3-O-β-D-glucopyranoside
	R1	R2
Askendoside C	β-D-ara(1→2)-β-D-xyl	H
Askendoside F	β-D-xyl(1→2)-β-D-ara	β-D-glc
Oleanane-type sapogenins
Soyasapogenol B			Sapogenin II

The earliest report on the chemical composition of the plant is on its flavonoid content. The species was reported to accumulate mainly flavonols (glycosides of kaempferol, quercetin, and isorhamnetin) as well as flavones (apigenin glycosides). Initially, cosmosin (apigenin-7-O-β-D-glucoside), astragalin (kaempferol-3-O-β-D-glucoside) and isorhamnetin-3-O-β-D-glucoside were identified in its aerial parts by paper chromatography with reference substances (Nikolov et al. 1984). Later in the eighties, kaempferol, quercetin, apigenin-7-О-arabinosyl-glucoside, kaempferol-3-О-xylosyl-glucoside, kaempferol-7-О-galactoside, and rutin were identified in the same plant parts by the same technique (Elenga 1986). In recent studies, kaempferol triglycosides have been reported in its aerial parts. The rare camelliaside A (kaempferol-3-O-[2-O-β-D-galactopyranosyl-6-O-α-L-rhamnopyranosyl]-β-D-glucopyranoside) was isolated for the second time from a plant source (Shkondrov et al. 2020a). In a total extract from its herbs, mauritianin (kaempferol-3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-galactopyranoside) was identified and quantified by ultra-high performance liquid chromatography-high resolution electrospray ionization mass spectrometry (UHPLC-MS) analysis using an authentic reference substance. The species could be considered a reliable source of this kaempferol triglycoside (Shkondrov et al. 2020b). Both camelliaside A and mauritianin occur rarely in plants, and their accumulation in this taxon is of future interest. The flavonoids from the species are presented in Table 2.

Table 2.

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Flavonoids in A. glycyphyllos.

R1	R2	R3	Flavonoid
H	O-β-D-glc	OH	Cosmosin
O-β-D-glc	OH	H	Astragalin
O-β-D-glc	OH	OCH3	Isorhamnetin-3-O-β-D-glucoside
OH	O-ara-glc	H	Apigenin-7-О-arabinosyl-glucoside
O-xyl-glc	OH	H	Kaempferol-3-О-xylosyl-glucoside
OH	O-gal	H	Kaempferol-7-О-galactoside
O-α-L-rha-β-D-glc	OH	OH	Rutin
O-[2-O-β-D-gal-6-O-α-L-rha]-β-D-glc	OH	H	Camelliaside A
O-α-L-rha-(1→2)-[α-L-rhal-(1→6)]-β-D-gal	OH	H	Mauritianin

The information on this group of secondary metabolites in this species is still quite limited. There are no reports on their presence in the roots, which are the usual source of these compounds. A mixture of sterols from the overground parts was obtained by column chromatography with Al₂O₃. Using their chromatographic characteristics, and the IR and MS spectra, the presence of ß-sitosterol, stigmasterol, and campesterol was elucidated (Elenga et al. 1986).

A complex study of four Astragalus species grown in Bulgaria was conducted. Among the samples, leaves, flowers, and fruits of A. glycyphyllos were investigated. Volatiles from the samples were obtained by steam distillation, and their chemical composition was studied by gas chromatography-mass spectrometry (GC-MS). The influence of the phenological stage on their chemical composition was examined as well. Hydrocarbons, as well as hydroxy-, aldoxy-, carboxy-, and other functional classes, were quantified. A significant change in the amount of hydrocarbons from leaf development to fructification was proven. It was notable that terpenes were not present in the fruit sample, unlike in the initial stages of leaf development, where these compounds were the predominant ones (Platikanov et al. 2005). The volatiles, proven in the plant are summarized in Table 3.

Initially, the total volatile compounds from the overground parts of the plant were tested for cytotoxic activity in vitro. REH cells (human acute lymphoid leukaemia) were exposed to various concentrations of the total volatiles (0.1, 1, 10, 100, and 1000 μg/ml) and a MTT test was carried out to evaluate the results. The tested extract induced a concentration-dependent inhibition of the proliferation of REH cells (Momekov et al. 2007).

Saponin-rich fractions produced from shoot cultures of A. glycyphyllos were tested on selected cell lines: T-24 (bladder carcinoma), CAL-29 (bladder transitional cell carcinoma), MJ (cutaneous T-cell lymphoma of the Mycosis fungoides type), and HUT-78 (cutaneous T-cell lymphoma of the Sézary syndrome type) via the standard MTT test. The saponin-rich fractions displayed high efficacy against urinary bladder cancer cells (IC₅₀ = 168.4 μg/mL) with a constitutive high expression level of the xenobiotic pump gp170 (MDR1) (Shkondrov et al. 2019). The antiproliferative effects of a total saponin mixture, a purified saponin fraction, and a cycloartane saponin (17(R),20(R)-3β,6α,16β-trihydroxycycloartanyl-23-carboxylic acid 16-lactone 3-O-β-D-glucopyranoside) from the aerial parts of a wild-grown plant were tested in different concentrations (12.5-200 µg/mL) against the same cell lines. The fraction had the strongest cytotoxic activity on the examined cell lines. This is considered a normal result of the activity of all of the saponins in it. Interestingly, the saponin mixture had a lower antiproliferative effect, which can be partially explained by its lower purity compared to the fraction obtained from it afterwards (Mihaylova et al. 2021).

A study to evaluate the possible in vitro and in vivo antiproliferative and cytotoxic activity of a purified saponin mixture (PSM) obtained from the species was also performed. The effects of PSM on the viability and proliferative activity on Graffi myeloid tumour cells were assessed by the MTT test. The cell viability values measured at two time intervals (on the 24^th and 48^th h) showed similar concentration-dependency. Moreover, no signs of reversibility of the cytotoxic action were proven. PSM induced a statistically significant decrease in cell viability (p < 0.01) compared to the control (non-treated Graffi tumour cells) on the 24^th hour at concentrations higher than 31 μg/mL. On the 48^th hour the values measured were slightly lower than those measured on the 24^th. The in vitro results provided the basis for an in vivo evaluation of the antiproliferative effects of the PSM in Graffi-tumour-bearing hamsters (GTBH). The effects of per-oral application of the PSM resulted in decreased tumour size and increased mean survival time. Pathohistological examination of the tumours of GTBH treated with PSM revealed a well-formed stroma with an accumulation of mononuclear cells and a lower quantity of mitotic cells, than in the GTBH from the non-treated control. It was also found that the mixture exerted tumour-protective effects. They were proved by a prolonged mean survival time (with four days more, compared to the non-treated GTBH) and a lowering in mortality rates, unlike the higher ones in the non-treated group of animals (Georgieva et al. 2021).

It is known that saponins possess immunomodulatory action, and some are even used as adjuvants in vaccines (Lacaille-Dubois 1999). The in vitro immune modulating activity of the cycloartane saponin 17(R),20(R)-3β,6α,16β-trihydroxycycloartanyl-23-carboxylic acid 16-lactone 3-O-β-D-glucopyranoside, obtained from the aerial parts of A. glycyphyllos, was examined on a murine model of isolate mice lymphocytes and peritoneal marcophages at different concentrations (1, 10, 20, 60, 100, 200 µmol/L). A statistically significant dose-dependent stimulation on both immune cell types (in concentrations above 10 μmol/L) was proved (Mihaylova et al. 2021).

The first in vivo investigation of A. glycyphyllos was performed on a model of CCl₄-induced liver damage in male Wistar rats. The levels of malonedialdehyde (MDA), reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST) as biochemical markers of oxidative stress in the liver were studied in a pool of 36 animals. The extract was administered orally in a dose of 100 mg/kg against a control group of animals treated with silymarin (100 mg/kg). The extract decreased MDA production by 24% and led to an increase in GSH levels by 36%, GPx activity by 83%, GR activity by 68%, and in GST activity by 51%. These values were comparable to those found in the control group (treated with silymarin at the same dose) (Shkondrov et al. 2015).

The polysaccharides, obtained from an n-butanol extract of the aerial parts of the species, were tested on isolated rat liver microsomes in a model of non-enzyme- and enzyme-induced lipid peroxidation (LPO). The polysaccharides were applied at concentrations of 0.6, 6, and 60 µg/mL on the microsomes to test their antioxidant properties. A significant reduction in MDA production was observed after incubation. This was a sign of concentration-dependent antioxidant activity. It was most pronounced at the highest concentration level and commensurable to that of the control substance (silymarin) (Kondeva-Burdina et al. 2016).

The neuroprotective effect of 17(R),20(R)-3β,6α,16β-trihydroxycycloartanyl-23-carboxylic acid 16-lactone 3-O-β-D-glucopyranoside was tested in different models of cyanotoxin (anatoxin-α)-induced neurotoxicity on subcellular fractions (rat brain microsomes, mitochondria, synaptosomes) in concentrations of 1, 10, and 100 μM. The saponin protected the synaptosomal viability and maintained the GSH level at 89% and 70%, respectively (at a concentration of 100 μM), compared with the pure anatoxin-α. Also, on isolated brain mitochondria, it preserved the GSH level and decreased MDA production, by 64% and by 48%. In conditions of anatoxin-α-induced toxicity on rat brain microsomes, the saponin (100 μM) decreased MDA production by 48% in comparison with the control (Ilieva et al. 2020). Further, the saponin, along with camelliaside A, were tested on a 6-hydroxydopamine (6-OHDA)-induced neurotoxicity model on isolated rat brain synaptosomes in vitro. Both compounds showed statistically significant neuroprotective activity at 100 μM. It showed activities comparable to those of silibinin, while camelliaside A had a weaker effect on synaptosomal viability (98% for the saponin and 32% for the flavonoid) against 6-OHDA). The levels of GSH were retained as follows: 56% for the saponin and 36% for the flavonoid against the toxic agent (Shkondrov et at. 2020a).

In another study, the same compounds (1 μM concentration) were also tested for possible activity on human recombinant monoamine oxidase type B enzyme (hMAO-B). They had an inhibitory effect, 44% and 35%, respectively, in comparison to the reference selegiline (55%). Thus, this saponin could be considered a promising structure in the treatment of neurodegenerative diseases (Shkondrov et al. 2020a).

A defatted extract from the plant (DEAG) was standardized in respect of saponins and tested for its antiviral activity in vitro alone and in combination with acyclovir against Simplexvirus humanalpha types 1 (acyclovir sensitive) and 2 (acyclovir resistant). DEAG was applied in concentrations ranging from 0.00625 mg/mL to 8 mg/mL and a maximal non-toxic concentration of 0.6 mg/mL was established. When used in that concentration, DEAG reached between 60% and 70% protection against both virus strains. The combination of the extract and acyclovir was tested against Simplexvirus humanalpha type 1 and led to antagonism, and it was concluded that they should not be used simultaneously (Shkondrov et al. 2023).