Flavonoids and saponins from two Bulgarian Astragalus species and their neuroprotective activity

Phytochemistry Letters 26 (2018) 44–49

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Flavonoids and saponins from two Bulgarian Astragalus species and their neuroprotective activity

T

⁎

Aleksandar Shkondrova, Ilina Krastevaa, , Franz Bucarb, Olaf Kunertc, Magdalena Kondeva-Burdinad, Iliana Ionkovaa a

Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Sofia, 2 Dunav St., 1000 Sofia, Bulgaria Department of Pharmacognosy, Institute of Pharmaceutical Sciences, University of Graz, Universitätsplatz 4, A-8010 Graz, Austria c Department of Pharmaceutical Chemistry, Institute of Pharmaceutical Sciences, University of Graz, Universitätsplatz 1, A-8010 Graz, Austria d Laboratory of Drug Metabolism and Drug Toxicity, Department of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Pharmacy, Medical University of Sofia, 2 Dunav St., 1000 Sofia, Bulgaria b

A R T I C LE I N FO

A B S T R A C T

Keywords: Astragalus glycyphylloides Astragalus spruneri Saponins Flavonoids Protective activity Synaptosomes

Two novel compounds from A. glycyphylloides DC (1–2) and three rare flavonoids from A. spruneri Boiss. (3–5), alongside their neuroprotective activity and possible effects as human monoamine oxidase type B inhibitors were reported. The structural elucidation of the compounds was achieved by chemical, HRESIMS and NMR analyses. Their effects were investigated in vitro on isolated rat brain synaptosomes in 6-hydroxydopamine-induced toxicity. In addition, their effect on human monoamine oxidase type B was explored, using Selegiline as a reference. 3-O-β-D-glucopyranosyl-28-O-[β-D-xylopyranosyl-(1 → 2)-β-D-glucopyranosyl] oleanolic acid (1) and kaempferol-3-O-β-D-glucopyranosyl-(1 → 4)-O-α-L-rhamnopyranosyl-(1 → 6)-O-[β-D-glucopyranosyl-(1 → 2)]-βD-galactopyranoside (2) were isolated for the first time, alongside three known flavonoids, baimaside (3), isobioquercetin (4) and quercetin-3-O-α-L-rhamnopyranosyl-(1 → 2)-[6-O-(3-hydroxy-3-methylglutaryl)-β-D-galactopyranoside (5). On 6-hydroxydopamine in vitro model in synaptosomes, compounds 1-3 had neuroprotective activity similar to that of Silybin A + B. All compounds exhibited weak activity on the monoamine oxidase type B enzyme.

1. Introduction The genus Astragalus (Fabaceae) is represented in the Bulgarian flora with 29 species (Valev, 1976). The plants have been reported to accumulate primarily flavonoids and saponins. Extracts and pure compounds from the genus have been demonstrated to possess immunomodulatory, antioxidant, hepatoprotective, and cardiovascular activities, among others (Bratkov et al., 2016; Ionkova et al., 2014; Krasteva et al., 2016). Astragalus glycyphylloides DC. (Pseudo Liquorice Milk-vetch) is a perennial, herbaceous flowering plant, native to Europe and commonly distributed in Bulgaria (Valev, 1976). Astragalus spruneri Boiss. (Spruner’s Milk-vetch) is a clump-forming perennial plant spread across the Balkan Peninsula and Turkey and belongs to the section Incani DC. of subgenus Cercidothrix Bunge (Valev, 1976). Thus, the phytochemical study of A. spruneri could help to find a chemotaxonomic marker for the section Incani DC. of this subgenus, where many species have

morphological similarities hard to distinguish (Krasteva et al., 2015). Ethanol extract from A. glycyphylloides had statistically significant cytoprotective and antioxidant activity in vivo and in vitro, similar to those of Silymarin, on carbon tetrachloride-induced cytotoxicity. Further phytochemical investigations of the extract led to the isolation of nine flavonoids: quercetin, quercetin-3-O-arabinoside, avicularin, hyperoside, isoquercitrin, kaempferol, isorhamnetin, isorhamnetin-3-Oglucoside, and isorhamnetin-3-O-arabinoside (Kondeva-Burdina et al., 2013; Simeonova et al., 2013). Up to date there are no reports on the chemical constituents of A. spruneri. Several studies have demonstrated the cytoprotective effects of flavonoids and saponins, isolated from Astragalus species (Bratkov et al., 2016; Ionkova et al., 2014; Krasteva et al., 2016). Moreover, oxidative stress leading to cell damage in the central nervous system is considered one of the leading factors in the pathophysiology of neurodegenerative disorders. In our previous studies (Kondeva-Burdina et al., 2014)

Abbreviations: hMAO, B human recombinant monoamine oxidase type B enzyme; HRESIMS, high resolution electro spray ionization mass spectrometry; NMR, nuclear magnetic resonance spectroscopy; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DTNB, 5,5′-dithio-bis-(2-nitrobenzoic acid); GSH, reduced glutathione ⁎ Corresponding author. E-mail address: [email protected] (I. Krasteva). https://doi.org/10.1016/j.phytol.2018.05.015 Received 27 March 2018; Received in revised form 2 May 2018; Accepted 4 May 2018 1874-3900/ © 2018 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

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Table 1 NMR spectroscopic data (1H 400 MHz, J in Hz)a and compound 1 (25 °C, TMS as internal standard).

flavonoid glycosides were reported to penetrate the blood-brain barrier. 13

C NMR (100 MHz) of

2. Results and discussion

1, pyridine-d5

2.1. Compounds from A. glycyphylloides position

δC, type

δH

1

38.8, CH2

2

26.6, CH2

3 4 5 6

88.9, 39.6, 55.9, 18.6,

7

33.2, CH2

8 9 10 11

39.8, 48.1, 37.0, 23.7,

12 13 14 15

122.7, CH 144.4, C 42.1, C 28.7, CH2

16

23.8, CH2

17 18 19

47.2, C 41.8, CH 46.3, CH2

20 21

30.8, C 34.1, CH2

22

32.4, CH2

23 24 25 26 27 28 29 30

28.3, CH3 17.0, CH3 15.5, CH3 17.5, CH3 26.2, CH3 176.5, C 33.1, CH3 23.7, CH3

0.90 1.41 1.80 2.22 3.38, – 0.77, 1.27 1.44 1.47 1.51 – 1.63 – 1.91 1.91 5.45 – – 1.27 2.28 1.90 1.97 – 3.19, 1.29 1.82 – 1.10 1.40 1.80 1.90 1.27, 0.94, 8.81, 1.05, 1.29, – 0.92, 0.91,

Glc-1 1 2 3 4 5 6

93.6, 80.3, 78.7, 70.8, 79.1, 62.1,

Xyl-2 1 2 3 4 5

105.9, CH 75.9, CH 78.5, CH 71.2, CH 67.4, CH2

5.46, d (7.4) 4.07 4.18 4.27 3.72, t (10.7) 4.40

Glc-3 1 2 3 4 5 6

106.7, CH 75.9, CH 78.8, CH 71.9, CH 78.3, CH 63.1, CH2

4.93, d (7.5) 4.06 4.27 4.23 4.03 4.42 4.58, dd (11.7, 2.2)

CH C CH CH2

C CH C CH2

CH CH CH CH CH CH2

Compound 1 was obtained as a white amorphous powder. The HRESIMS spectrum showed a formic acid adduct [M+HCOO]− at m/ z = 957.5071 (calcd. 957.5059). A molecular formula of C47H76O17 was established by the MS and 13C NMR data. GC–MS analysis of the sugars indicated D-glucose and D-xylose. The NMR data of compound 1 (Table 1), especially the presence of seven methyl signals at δН 1.27(s), 0.94 (s), 8.81 (s), 1.05 (s), 1.29 (s), 0.92 (s) and 0.91 (s) in the proton spectrum, a signal of an olefin proton at δН 5.45, as well as for H-18 β at δН 3.19 (dd, J = 13.5, 4.1) and three anomeric methine groups in the HSQC spectrum, suggested the presence of an oleanane-derived triterpene triglycoside (Seebacher et al., 2003). A signal corresponding to Н-3 (δН 3.38, dd, J = 11.7, 4.5) was observed. From the 13С-NMR of 1 two resonances (δС 122.7 and δС 144.4) of С-12 and С-13 from the vinyl part of Δ12-oleanane were recorded. The signal at δС 88.9 corresponded to С-3 substituted with an OH group. The chemical shift for С-17 (δС 180.38) indicated the presence of carboxylic function at this position. By comparison with reference data the aglycone was confirmed as oleanolic acid, with chemical shifts changed due to attachment of sugar moieties at C-3 and C-28 (Seebacher et al., 2003). The sugar moieties were identified after complete assignments of proton and carbon data as two glucose and one xylose pyranoses in the β-form (Table 1). The anomeric proton (δH 6.22) of the glucose (Glc-1) showed a three bond HMBC correlation to C-28 (δC 176.5) of the aglycone, while an HMBC correlation between the anomeric proton (δH 4.93) of the glucose moiety (Glc-3) and C-3 (δC 88.9) of the oleanolic acid indicated a bisdesmosidic saponin. The anomeric proton (δH 5.46) of the xylose showed a HMBC correlation to C-2 (δC 80.3) of the glucose unit (Glc-1). Hence, compound 1 was identified as 3-O-β-D-glucopyranosyl-28-O-[βD-xylopyranosyl-(1 → 2)-β-D-glucopyranosyl] oleanolic acid (Fig. 1). Compound 2 was obtained as a yellow amorphous powder. The HRESIMS spectrum showed a quasi-molecular ion [M-H]− at m/ z = 917.2573 (calcd. 917.2563), indicating a molecular formula of C39H50O25, confirmed by 13C NMR data. The GC–MS analysis of the sugar chain showed D-galactose, L-rhamnose and D-glucose. In the 1H NMR spectrum of 2 (Table 2) signals for four protons from a kaempferol moiety, i.e. δH 6.72, d, J = 2.1 (H-6), δH 6.70, d, J = 2.1 (H-8), δH 8.48, d, J = 8.8 (H-2′ and Н-6′) and δH 7.25, d, J = 8.8 (H-3′ and Н-5′) were observed (Markham, 1982). The 13C NMR revealed a signal of carbonyl atom at δC 178.9 (C-4) and four signals corresponding to hydroxylated carbon atoms at δC 165.7 (C-7), δC 162.9 (C-5), δC 161.6 (C-4′) and δC 134.3 (C-3). The values of the chemical shifts for C-3 and C-2 (Table 2) suggested glycosylation of the C-3 OH-group (Markham, 1982). The data from the NMR analysis confirmed the presence of four hexose moieties, one of which a 6-desoxy sugar (δH 1.51, d, J = 6.0), and two of them with terminal hydroxymethylene groups. After complete assignments of proton and carbon NMR resonances in the 1D and 2D NMR experiments, the four hexoses were identified as one β-galactopyranose, two β-glucopyranoses, and one α-rhamnopyranose. The anomeric proton (δH 6.51) of the galactose showed a three bond correlation to C-3 (δC 134) of the aglycone, the anomeric proton (δH 5.45) of the glucose (Glc-2) to C-2 (δC 82.2) of the galactose moiety, the anomeric proton (δH 5.45) of the rhamnose to C-6 (δC 65.9) of the galactose unit, and the anomeric proton (δH 5.12) of the glucose (Glc-4) to C-4 (δC 85.3) of the rhamnose. Compound 2 was thus assigned as kaempferol-3-O-β-D-glucopyranosyl-(1 → 4)-O-α-L-rhamnopyranosyl-(1 → 6)-O-[β-D-glucopyranosyl-(1 → 2)]-β-D-galactopyranoside (Fig. 1).

dd (11.7, 4.5) d (12.7)

dd (13.5, 4.1)

s s s s s s s

6.22, d (7.6) 4.33 4.30 4.31 3.93 4.35 4.41

Multiplicity of obscured signals is not labelled, chemical shift values from HSQC. a The assignments were based on 1D 1H, 13C and 2D DQF-COSY, HSQC, and HMBC experiments.

2.2. Flavonoids from A. spruneri Flavonoids 3-5 were identified by acid hydrolysis, UV spectroscopy, 45

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Fig. 1. Structures of compounds from A. glycyphylloides.

Table 2 NMR spectroscopic data (1H 400 MHz, J in Hz)a and compound 2 (25 °C, TMS as internal standard).

13

of Rosa (Porter et al., 2012). As already suggested, their presence in Astragalus species for establishing the taxonomic structure of section Incani DC. could be significant (Krasteva et al., 2015).

C NMR (100 MHz) of

2, pyridine-d5

2.3. Activity on isolated synaptosomes position

δC, type

δH

position

δC, type

δH

2 3 4 4a 5 6 7

156.9, C 134.3, C 178.9, C 105.3, C 162.9, C 99.8, CH 165.7, C

– – – – – 6.72, d (2.1) –

Gal-1 1 2 3 4 5 6

100.3, CH 82.2, CH 75.2, CH 68.9, CH 74.9, CH 65.9, CH2

6.51, d (7.6) 4.86 4.29 4.37, s 4.04 3.73 4.34

8 8a 1′ 2′/6′ 3′/5′ 4

94.5, CH 157.5, C 122.0, C 132.0, CH 116.2, CH 161.6, C

6.70, d (2.1) – – 8.48, d (8.8) 7.25, d (8.8) –

Glc-2 1 2 3 4 5 6

106.1, CH 75.9, CH 78.5, CH 71.4, CH 78.7, CH 62.5, CH2

5.45, d (7.4) 4.21 4.17 4.26 3.83 4.32 4.42

Rha-3 1 2 3 4 5

101.4, CH 71.5, CH 72.6, CH 85.3, CH 67.9, CH 18.3, CH3

5.07, brs 4.35 4.42 4.23 4.17 1.51, d (6.0)

Glc-4 1 2 3 4 5 6

106.9; CH 76.4; CH 78.3; CH 71.4; CH 78.4; CH 62.5, CH2

5.12, d (7.7) 4.04 4.21 4.26 3.73 4.32 4.42

Administered alone, 6-hydroxydopamine (6-OHDA, 150 μM) revealed a statistically significant neurotoxic effect on isolated rat brain synaptosomes by decreasing synaptosomal viability and level of reduced glutathione (GSH), compared to the control (non-treated synaptosomes, 100% viability) (Figs. 2 and 3). In conditions of 6-OHDAinduced oxidative stress (150 μM), the pre-treatment of the synaptosomes with the plant compounds (100 μM) lead to the statistically significant neuroprotective and antioxidant activity, compared to the toxic agent (6-OHDA). Toxic effects of 6-OHDA were assumed as 100%. The effects of the compounds observed were referred to those of the toxic agent. Compounds 1, 2 and 3 showed neuroprotective and antioxidant effects similar to those of Silybin A + B (Silibinin, 100 μM) – one of the major components of Silymarin. Flavonoids 4 and 5 revealed neuroprotective and antioxidant effects, but they are weaker than those of Silybin, saponin 1 and flavonoids 2 and 3. 2.4. Activity on hMAO-B The isolated compounds (1-5) had lower statistically significant inhibitor activity (1 μM) on human recombinant MAO-B (hMAO-B) enzyme than Selegiline (1 μM)(Fig. 4), compared to the control (pure hMAO-B). This could lead to the suggestion that the above-mentioned protective effects on synaptosomes in a model of 6-OHDA-induced oxidative stress could not be a direct result of hMAO-B inhibition, but of free radical scavenging capabilities of the investigated compounds.

The assignments were based on 1D 1H, 13C and 2D DQF-COSY, HSQC, and HMBC experiments. Multiplicity of obscured signals is not labelled, chemical shift values from HSQC.

3. Experimental

HRESIMS, complete assignments of their 1H and 13C NMR and by comparison with literature data as: quercetin-3-O-β-D-glucopyranosyl(1 → 2)-β-D-glucopyranoside (baimaside, 3) (Gluchoff-Fiasson et al., 1997), quercetin-3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-galactopyranoside (isobioquercetin, 4) (Yasukawa et al., 1990), and quercetin-3-O-αL-rhamnopyranosyl-(1 → 2)-[6-O-(3-hydroxy-3-methylglutaryl)-β-D-galactopyranoside] (5) (Porter et al., 2012). This is the first report on the flavonoid content of A. spruneri. For the second time a flavonol, acylated with 3-hydroxy-3-methylglutaric acid was isolated from species of section Incani DC. (subgenus Cercidothrix Bunge) of this genus. These type of flavonoids were considered only as a chemotaxonomic marker

UV spectra were recorded in absolute MeOH on a UV–vis Libra S70 spectrophotometer (Biochrom, United Kingdom). NMR spectra (1D 1H, 13 C, 2D DQFCOSY, HSQC and HMBC) were recorded on a Varian UnityInova spectrometer operating at a proton frequency of 400 MHz. All compounds were dissolved in 0.72 mL pyridine-d5 with 0.1% TMS as an internal standard. Optical rotations were measured on AUTOPOL VI Automatic Polarimeter (Rudolph Research Analytical, USA). The 1D 1H, 13 C and 2D DQF-COSY, HSQC, and HMBC experiments were performed at 25 °C with standard pulse programs from the Varian user library. Accurate mass determinations were performed using a LC/FTMS system consisting of an Exactive Orbitrap mass spectrometer, equipped with a HESI source (ThermoFisher Scientific, Inc., Bremen, Germany) and

a

3.1. General experimental procedures

46

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Fig 2. Effects of the plant compounds (1–5) and Silybin (100 μM) on synaptosomal viability, in conditions of 6-OHDA-induced oxidative stress in rat brain synaptosomes; *** p < 0.001 vs control (non-treated synaptosomes); +p < 0.05; +++p < 0.001 vs 6-OHDA.

(LPLC). Semi-preparative HPLC was performed on a Waters® (Milford MA, USA) system consisting of a binary gradient pump (model 1525EF), manual injector (7725i), UV detector (model 2489) and Breeze software v. 2. A semi-preparative ODS column Luna® (100 Å, 250 × 10 mm, 5 μm, Phenomenex, Torrance CA, USA) was used and elution was performed with H2O-o-H3PO4 0.05% – MeCN at 4.5 mL/min. The acid was then removed from the pure compound by solid phase extraction. All chromatograms were monitored at 204 nm (saponins) or at 254 and 330 nm (flavonoids). GC–MS analysis of (2R)-2-butyl glycosides was performed using an Exactive Orbitrap GC–MS system (ThermoFisher Scientific, Inc., Bremen, Germany) operating at 70 eV, ion source temperature 230 °C, interface temperature 280 °C. A split injection (1 μL injection volume, split ratio, 20:1) at 270 °C injector temperature was utilized. A fused silica capillary column, 5% phenyl/95% methyl polysiloxane (HP-5MS 30 m x 250 μm × 0.25 μm, Agilent J & W, USA), was used. The temperature program was as follows: 100 °C, then 270 °C at 3 °C/ min. The carrier gas was helium 5.6 at a flow rate of 1.4 mL/ min. Data acquisition was performed with Xcalibur version 4 for the mass scan range 40–600 u.

operated in ultra-high resolution mode (100.000) coupled to a U-HPLC system (Dionex, ThermoFisher Scientific, Inc., Bremen, Germany). Operating conditions for the ESI source used in the negative ionization mode were: 4.6 kV spray voltage, 250 °C capillary temperature, sheath gas flow rate 50 units and auxiliary gas flow 5 units (units refer to arbitrary values set by the Exactive software). Nitrogen was used for sample nebulization. U-HPLC separations were performed on a Hypersil Gold C18 (ThermoFisher Scientific, USA), 1.9 μm, 2.1 × 50 mm i.d. HPLC column, operated at 30 °C. Each 10 min chromatographic run was carried out at a flow rate of 0.3 mL/min with a binary mobile phase consisting of MeOH + 0.1% HCOOH (A) and 10 mM NH4COOH + 0.1% HCOOH (B) using a gradient of 50% A for 0.5 min, up to 100% A in 5 min, isocratic at 100% for 0.5 min, then back to 50% A for 0.1 min. TLC was carried out on precoated silica gel plates (Kieselgel® G, F254, 60, Merck, Darmstadt, Germany) with EtOAc-HCOOH-H2O (10:1:4), EtOAc-HCOOH-AcOH-H2O (32:3:2:6) and EtOAc-EtCOMe-HCOOH-H2O (5:3:1:1). Plates were sprayed with anisaldehyde/conc. H2SO4 and heated for 10 min at 104 °C (saponins) or under UV light (365 nm) by spraying with Naturstoff Reagenz A (1% solution of 2-aminoethyl ester of diphenyl boric acid) and over spraying with 5% of methanol solution of polyethylene glycol 4000 (flavonoids). Column chromatography (CC) was performed using Diaion® HP-20 (Supelco, USA) and Sephadex® LH20 (Pharmacia Fine Chemicals AB, Uppsala, Sweden). Silica gel (MN Kieselgel® 60 0.04-0.063 mm, Macherey-Nagel, Düren, Germany) and Octadecylsilyl (ODS) gel DAVISIL® (Grace Davison Discovery Sciences, Hesperia CA, USA) were used for low pressure liquid chromatography

3.2. Plant material The aerial parts of A. glycyphylloides DC were collected in July 2012 from Rila Mountain, Bulgaria. The over ground parts of A. spruneri Boiss. were collected in April 2014 from Kozhuh Mountain, Rupite area, Bulgaria. The plants were identified by Dr. D. Pavlova from the Department of Botany, Faculty of Biology, Sofia University, where

Fig. 3. Effects of the plant compounds (1–5) and Silybin (100 μM) on GSH level, in conditions of 6-OHDA-induced oxidative stress in rat brain synaptosomes; ** p < 0.01 vs control (non-treated synaptosomes);+p < 0.05; ++ p < 0.01 vs 6-OHDA. 47

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Fig. 4. Effects of the plant compounds (1–5) and Selegiline (1 μM) on hMAO-B activity; ** p < 0.05; *** p < 0.001 vs control (pure hMAOB).

3.4. 3-O-β-D-glycopyranosyl-28-O-[β-D-xylopyranosyl-(1 → 2)-β-Dglycopyranosyl] oleanolic acid (1)

voucher specimens have been deposited: SO-093817 (A. glycyphylloides DC.) and SO-107625 (A. spruneri Boiss.).

White amorphous powder; C47H76O17; [α]D 20 = +3.764 (c 0.1, MeOH); HREIMS m/z 957.5071 [M+HCOO]− (calcd. for C48H76O19, 957.5059); 1H NMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz), see Table 1.

3.3. Extraction and isolation The air-dried powdered plant material from A. glycyphylloides (350 g) was exhaustively extracted with 80% MeOH (15 × 1 L) on an ultrasonic bath (35 kHz, 1 h each). The extract was filtered and concentrated. The hydrophilic residue was suspended in water and exhaustively extracted with CH2Cl2 to remove the lipophilic constituents. The defatted water residue was successively extracted with EtOAc and n-BuOH. The n-BuOH extract was evaporated to dryness to give a solid residue (12.3 g) and then it was subjected to CC over Diaion HP-20 (4.7 × 45 cm), eluting with H2O-MeOH (100:0 → 0:100, v/v) to give nine main fractions (I–IX). Fraction II was chromatographed over Sephadex LH-20, eluting with MeOH, and 6 sub fractions were collected (A1-A6). Sub fraction A3 was subjected to LPLC over an ODS column (2.4 × 30 cm), eluting with MeOH-H2O (28:72, v/v) to give four sub fractions (B1-B4). Sub fraction B1 was further subjected to LPLC over an ODS column (1.5 × 15 cm) eluting with MeOH-H2O (23:77, v/v) and purified by semi-preparative HPLC with MeOH-H2O (12:88, v/v) to give compound 2 (3.5 mg). Fraction VII was chromatographed over a silica gel column, eluting with a gradient of CH2Cl2-MeOH-H2O (90:10:0 → 80:20:3, v/v/v) to obtain 37 fractions (C1-C37). Fraction C5 was purified by gel filtration over a Sephadex LH-20 column, eluting with MeOH and three sub fractions were collected (D1-D3). Sub fraction D2 was further subjected to CC over a silica gel column, eluting with a gradient of CH2Cl2-MeOH-H2O (90:10:0 → 80:20:3, v/v/v) and afterwards over Sephadex LH-20 column with MeOH as eluent to obtain compound 1 (8.2 mg). The air-dried plant material of A. spruneri (200 g) was subjected to extraction with CH2Cl2 to remove the lipophilic constituents. The defatted plant material was exhaustively extracted with a gradient of MeOH-H2O (100:0 → 80:20, v/v) by percolation. The extract was filtered and the solvent was removed under reduced pressure. The water residue was successively extracted with EtOAc and n-BuOH. The nBuOH extract was evaporated to dryness to give a solid residue (9 g) and then it was subjected to CC over Diaion HP-20 (4.7 × 45 cm), eluting with H2O-MeOH (100:0 → 0:100, v/v) to give five main fractions (I–V). Fraction II was subjected to CC over Sephadex LH-20 eluted with MeOH and four sub fractions were collected (G1-G4). Sub fraction G2 was chromatographed by LPLC over ODS column eluting with MeOH-H2O (25:75, v/v) to give four subtractions (H1-H4). Sub fraction H3 was separated by isocratic semi-preparative HPLC, eluting with a mobile phase of MeCN-H2O (17:83, v/v) to give compounds 3 (6 mg), 4 (18 mg) and 5 (7 mg).

3.5. Kaempferol-3-O-β-D-glucopyranosyl-(1 → 4)-O-α-L-rhamnopyranosyl(1 → 6)-O-[β-D-glucopyranosyl-(1 → 2)]-β-D-galactopyranoside (2) Yellow amorphous powder; C39H50O25; UV (MeOH) λmax (log ε) 259 (4.05), 293 (sh) (3.82), 345 (3.99) nm; [α]D 20 = −64.282 (c 0.1, MeOH); HREIMS m/z 917.2573 [M-H]− (calcd. for C39H49O25, 917.2563); 1H NMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz), see Table 2.

3.6. Determination of absolute configuration of sugars The analysis was performed by a GC–MS method (Reznicek et al., 1993) including acidic hydrolysis and preparation of (2R)-2-butyl glycosides. Standard compounds L-rhamnose, D-glucose, D-xylose and Dgalactose (Sigma-Aldrich, Germany) were treated by the same protocol. The (2R)-2-butylglycosides were analysed by GC–MS, obtaining peaks at tR = 20.74, 22.35 (L-rha), 27.62, 28.53 (D-xyl), 28.80, 29.71 (D-gal), and 29.87, 31.94 (D-glc) min.

3.7. Rat brain synaptosomes and hMAO-B assay Synaptosomes were prepared by brains from old male Wistar rats (Taupin et al., 1994). The content of synaptosomal protein was determined using serum albumin as a standard (Lowry et al., 1951). Synaptosomal viability was measured by MTT-test (Mungarro-Menchaca et al., 2002). Level of GSH in synaptosomes was determined (Robyt et al., 1971) with the Ellman reagent (DTNB). All tested substances were removed after the incubation period via centrifugation to eliminate interference with the colorimetric methods. For the hMAO-B assay a fluorimetric method which includes Amplex UltraRed reagent (Zhou et al., 1997) was used. To test the ability of the substances to modify fluorescence during the course of the reaction (non-enzymatic interaction), solutions of the test substances and the Amplex UltraRed reagent in phosphate buffer were mixed. Blank samples were prepared without a substrate (Bautista-Aguilera et al., 2014). 48

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4. Conclusion

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From the aerial parts of A. glycyphylloides a novel pentacyclic saponin (1) and a new flavonol tetraglycoside (2) were isolated. Three rare flavonoids (3–5) were elucidated from the aboveground parts of A. spruneri. From the conducted in vitro study on rat synaptosomes we conclude that saponin 1 and flavonoids 2 and 3 displayed neuroprotective and antioxidant activities, comparable to that of Silybin A + B. In addition, flavonoids 4 and 5 showed lower potential as possible antioxidants and neuroprotectors, compared to the latter. On hMAO-B enzyme all compounds 1-5 showed weaker inhibitory activity than Selegiline. Conflict of interest Authors declare no competing interest. Acknowledgments This work was supported by National Scientific Fund at Ministry of Education and Science of Republic of Bulgaria, contract № D H03/6/ 17.12.2016 and a Grant Project № 7765/22.11.2017 from Medical Science Council at Medical University of Sofia. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.phytol.2018.05.015. References Bautista-Aguilera, O.M., Esteban, G., Bolea, I., Nikolic, K., Agbaba, D., Moraleda, I., Iriepa, I., Samadi, A., Soriano, E., Unzeta, M., et al., 2014. Design, synthesis, pharmacological evaluation, QSAR analysis, molecular modeling and ADMET of novel donepezil–indolyl hybrids as multipotent cholinesterase/monoamine oxidase inhibitors for the potential treatment of Alzheimer’s disease. Eur. J. Med. Chem. 75, 82–95. Bratkov, V., Shkondrov, A., Zdraveva, P., Krasteva, I., 2016. Flavonoids from the genus Astragalus: phytochemistry and biological activity. Pharmacogn. Rev. 10, 11. http:// dx.doi.org/10.4103/0973-7847.176550. Gluchoff-Fiasson, K., Fiasson, J.L., Waton, H., 1997. Quercetin glycosides from European

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