Flavoniods from aerial parts of Astragalus hoantchy

Fitoterapia 114 (2016) 34–39

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Flavoniods from aerial parts of Astragalus hoantchy Kai Guo a, Xiaofeng He a, Yanping Zhang b, Xiuzhuang Li a, Zhiqiang Yan a, Le Pan a, Bo Qin a,⁎ a Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China b Fisheries Research Institute of Gansu Province, Lanzhou 730030, PR China

a r t i c l e

i n f o

Article history: Received 14 June 2016 Received in revised form 17 August 2016 Accepted 20 August 2016 Available online 23 August 2016 Keywords: Astragalus hoantchy Leguminosae Flavonoids Antifungal activity

a b s t r a c t Four new flavonoids, astragaisoflavans A–D (1–4), along with thirteen known ones (5–17) were isolated from the aerial parts of Astragalus hoantchy. Their structures were established by the extensive spectroscopic analyses including one- and two-dimensional NMR, HRESIMS, IR, and UV results. The absolute configurations of 1–4 were deduced based on circular dichroism. Compounds 1–3 all possessed a rare modified A-ring and compound 4 was a dimeric isoflavan. Compound 5 was first identified from genus Astragalus and 6–17 were found from A. hoantchy for the first time. Compound 1 and 4 were evaluated for their antifungal activity against Alternaria solani (a phytopathogenic fungus), with the result that 1 failed to exhibit significant antifungal activity at the testing concentration of 100 μg/mL and the IC50 value of 4 was 173.3 μg/mL. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Flavonoids, a group of ubiquitous and diverse secondary metabolites in higher plants, play a vital role in plants including defensing UV damage and phytopathogens, serving as pigments of flower, modulating auxin distribution, and acting as signal molecules towards symbiotic microbes [1–3]. Importantly, flavonoids have been reported to exhibit a variety of biological activities such as scavenging free radicals, remedying cardiovascular disease, anti-tumor, anti-microbial and antidiabetes [4–6]. Astragalus hoantchy, a perennial plant of the genus Astragalus (Leguminosae), is mainly distributed in north and northwest of China [7]. Its roots are used as traditional Chinese medicine (the substitute of prescribed Radix Astragali) and Mongolian medicine, for general debility, chronic illnesses, increasing overall vitality, and adjunct cancer therapy [8–10]. A few studies show that the chemical constituent of the root of A. hoantchy primarily contains flavonoids, triterpenoids and their glycosides, while the phytochemicals of its aerial parts have not been reported [11,12]. In our search for unique and bioactive compounds from this plant, four new and thirteen known flavonoids were isolated from the aerial parts of Astragalus hoantchy (Fig. 1). Among them, compounds 1–3 with the isoflavan skeleton all possessed a rare modified A-ring and compound 4 was a dimeric isoflavan. Compound 5 was first isolated from the genus Astragalus and 6–17 were found from A. hoantchy for the first time. Herein, we reported the isolation and structural

⁎ Corresponding author. E-mail address: [email protected] (B. Qin).

http://dx.doi.org/10.1016/j.fitote.2016.08.009 0367-326X/© 2016 Elsevier B.V. All rights reserved.

elucidation of new compounds (1–4), as well as the antifungal activity evaluation of selected compounds (1 and 4) against Alternaria solani. 2. Experimental 2.1. General IR spectra were measured on a Nicolet NEXUS 870 FT-IR spectrometer. UV spectra were recorded using a Shimadzu UV-1750 spectrophotometer. Optical rotations were obtained on a Perkin-Elmer 341 digital polarimeter. CD spectra were performed using an Olis DSM 1000 spectrometer. HRESIMS data were measured on a Bruker micoTOF-QII mass spectrometer. 1D and 2D NMR spectra were recorded using a Bruker AM-400BB instrument with TMS as internal standard. Column chromatography (CC) was carried out on silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Sephadex LH-20 (25–100 μm, Pharmacia Fine Chemical Co., Ltd., Berlin, Germany), and MCI gel (75–150 μm, Mitsubishi Chemical Industries, Ltd.). Standard and preparative thin-layer chromatography (TLC and PTLC) were performed on silica gel (GF254, 10–40 μm; Qingdao Haiyang Chemical Co., Ltd.). Spots were detected on TLC under UV light or by heating after spraying with 5% H2SO4 in C2H5OH (v/v). 2.2. Plant material The aerial parts of A. hoantchy were collected in October 2014 in Anyang, Gansu Province, China. The plant was identified by Professor Huanyang Qi from Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences (CAS). A voucher specimen (Ah-P-01) was deposited in Lanzhou Institute of Chemical Physics, CAS.

K. Guo et al. / Fitoterapia 114 (2016) 34–39

1

O

9

1

O

7

H 5

OH

3

O

OH

9

O

7

H

OCH3

1' 3'

5

OH

1'

3

3'

OH

1''

2'''

3

OH OCH3

1' 3'

OCH3 3

O H 3

5'

1'

H

OCH3

5

5''

HO

OCH3 5 R1

O R2

O R1

O

R2

O H

R3 R4

OCH3 OCH3

R3

OH O

8 R1=R2=R3=H 9 R1=R3=H, R2=OH 10 R1=R2=OH, R3=H

6 R=H 7 R=Glc

OCH3

OH

3'

OH

OR

OH

OH H3CO

OH

OH

O

O

7

7''

1''' 3''

9

4

H3CO

OH

H

1

O

9''

H

H3CO

5

1

2 O

5'''

9

7

OCH3 OCH3

1

OCH3 O

O

OCH3

H3CO

35

11 R1=R3=OH, R2=OCH3, R4=H 12 R1=H, R2=R3=OH, R4=OCH3 13 R1=H, R2=R4=OH, R3=OCH3

R OH

OH HO

O

HO

H O

HO

OCH3

OH O 14 R=H 15 R=OCH3

OCH3 OH

16

O H O 17

Structures of compounds 1–17 from the aerial parts of A. hoantchy Fig. 1. Structures of compounds 1–17 from the aerial parts of A. hoantchy.

2.3. Extraction and isolation The air-dried aerial parts of A. hoantchy (12.4 kg) were smashed and extracted with 95% EtOH-H2O (40 L) for five times (five days each time) at room temperature, then the combined solution was evaporated under reduced pressure to obtain the residue (1.2 kg), which was dissolved in distilled water and extracted in turn with petroleum ether (PE), EtOAc, and n-BuOH. The PE partition (255 g) was subjected to silica gel CC eluted with PE/Me2CO step gradient system (50:1, 20:1, 10:1, 5:1, 3:1, 1:1, and 0:1 v/v) and finally washed with MeOH to give eight fractions (Fr.1–Fr.8). Fr.5 (12 g) was applied to silica gel CC eluted with PE/EtOAc step gradient system (3:1, 2:1, and 1:1 v/v) to get three fractions (Fr.5-1–Fr.5-3). Fr.5-1 (4 g) was fractionated on silica gel CC with PE/EtOAc (5:1) as the mobile phase to obtain four fractions (Fr.5-1-1–Fr.5-1-4). Fr.51-3 (400 mg) was subjected to silica gel CC eluted with CHCl3/EtOAc (40:1) and submitted to PTLC to yield 13 (1.5 mg). Fr.6 (15 g) was applied to silica gel CC with CHCl3/EtOAc (3:1) as the mobile phase to give four fractions (Fr.6-1–Fr.6-4). Fr.6-1 (120 mg) was separated on Sephadex LH-20 eluted with CHCl3/MeOH (1:1) to yield 11 (43 mg). Fr.6-2 (560 mg) was separated on Sephadex LH-20 eluted with CHCl3/ MeOH (1:1) to get three fractions (Fr.6-2-1–Fr.6-2-3). Fr.6-2-1 (180 mg) was purified by silica gel CC eluted with CHCl3/EtOAc (7:1) to yield 12 (6 mg) and 8 (8.5 mg), respectively. Fr.6-2-2 (75 mg) was

fractionated on silica gel CC with CHCl3/EtOAc (3:1) as the mobile phase and submitted to PTLC to yield 2 (4 mg) and 1 (23 mg), respectively. Fr.7 (11 g) was applied to silica gel CC eluted with CHCl3/MeOH (15:1) to give three fractions (Fr.7-1–Fr.7-3). Fr.7-1 (6.5 g) was subjected to MCI gel CC eluted with H2O/MeOH step gradient system (1:0, 1:1, 1:3, 1:7, and 0:1 v/v) to produce five fractions (Fr.7-1-1–Fr.7-1-5). Fr.71-1 (200 mg) was purified by silica gel CC eluted with CHCl3/Me2CO (6:1) to obtain four fractions (Fr.7-1-1-1–Fr.7-1-1-4). Fr.7-1-1-1 (80 mg) was fractionated on silica gel CC with CHCl3/EtOAc (5:1) as the mobile phase and submitted to PTLC to yield 16 (5 mg) and 3 (3.5 mg), respectively. Fr.7-1-1-2 (30 mg) and Fr.7-1-1-3 (43 mg) were respectively submitted to PTLC to yield 17 (11 mg) and 5 (22 mg). Fr.8 (10 g) was subjected to MCI gel CC eluted with H2O/ MeOH step gradient system (1:0, 1:1, 1:3, 1:7, and 0:1 v/v) to produce five fractions (Fr.8-1–Fr.8-5). Fr.8-3 (120 mg) was purified by silica gel CC eluted with CHCl3/MeOH (7:1) to yield 7 (25 mg). The EtOAc partition (190 g) was subjected to silica gel CC eluted with CHCl3/MeOH step gradient system (100:1, 50:1, 25:1, 10:1, and 0:1 v/v) to give five fractions (Fr.e1–Fr.e5). Fr.e2 (4 g) was applied to silica gel CC eluted with CHCl3/MeOH (40:1) to produce three fractions (Fr.e2-1– Fr.e2-3). Fr.e2-2 (260 mg) was separated on Sephadex LH-20 eluted with CHCl3/MeOH (1:1) and purified by silica gel CC eluted with CHCl3/MeOH (30:1) to yield 15 (28 mg). Fr.e3 (8 g) was subjected to silica gel CC eluted with CHCl3/MeOH (30:1) to produce three fractions

36

K. Guo et al. / Fitoterapia 114 (2016) 34–39

2.3.4. Astragaisoflavan D (4) Amorphous yellow solid; [α]20 D –40 (c 0.4, MeOH); IR (neat) νmax 3400, 2937, 1597, 1502, 1458, 1421, 1155, 1110, 1022 cm−1; UV (MeOH) λmax (log ε) 227 (2.984), 283 (1.073) nm; CD (MeOH, Δε) λmax 328 (+ 9.57), 238 (+ 11.1), 231 (− 9.26), 223 (+ 29.7) nm; 1H and 13C NMR data, see Table 2; HRESIMS m/z 625.2044 [M + Na]+ (calc. for C34H34O10 + Na, 625.2050).

(Fr.e3-1–Fr.e3-3). Fr.e3-1 (140 mg) was separated on Sephadex LH-20 eluted with CHCl3/MeOH (1:1) and purified by silica gel CC eluted with CHCl3/MeOH (25:1) to yield 10 (10 mg). Fr.e4 (12 g) was fractionated on silica gel CC eluted with CHCl3/MeOH (8:1) to give five fractions (Fr.e4-1–Fr.e4-5). Fr.e4-1 (1.2 g) was separated on Sephadex LH-20 eluted with CHCl3/MeOH (1:1) to get three fractions (Fr.e4-1-1–Fr.e41-3). Fr.e4-1-1 (480 mg) was separated on Sephadex LH-20 eluted with CHCl3/MeOH (1:1) and purified by silica gel CC eluted with CHCl3/EtOAc (2.5:1) to yield 4 (24 mg). Fr.e4-3 (640 mg) was subjected to silica gel CC eluted with CHCl3/EtOAc (2:1) and separated on Sephadex LH-20 eluted with CHCl3/MeOH (1:1) to yield 9 (150 mg). Fr.e4-4 (350 mg) was separated on Sephadex LH-20 eluted with CHCl3/MeOH (1:1) and purified by silica gel CC eluted with CHCl3/ EtOAc (10:1) to yield 6 (45 mg). Fr.e4-5 (150 mg) was separated on Sephadex LH-20 eluted with CHCl3/MeOH (1:1) to yield 14 (25 mg).

2.4. Antifungal activity assay [13] Alternaria solani (A. solani, a phytopathogenic fungus, could cause the destructive disease named “early blight” in tomatoes and potatoes) was applied to evaluate the antifungal activity of selected compound 1 (compound 1, 2 and 3 had the same molecular skeleton, while 1 was the most abundant) and 4 in vitro by measuring the radial growth of mycelia. The compounds were dissolved in DMSO as stock solutions, which were then diluted with potato dextrose agar (PDA) medium to a testing concentration of 100 μg/mL and poured into the sterilized Petri dishes (diameter: 6 cm). After solidification, a mycelia disk (diameter: 0.6 cm) was inoculated in the center of each dish, which was then sealed with parafilm and incubated at 25 °C in dark for 4 days. The same volume of DMSO was added to PDA medium as control and three replicates were carried out to every measurement. Compound 4 was further tested under concentrations (200, 100, 50 and 25 μg/mL) to calculate the IC50 value [14].

2.3.1. Astragaisoflavan A (1) Colorless needle crystal; [α]20 D + 42 (c 0.8, MeOH); IR (neat) νmax 3467, 2935, 1665, 1601, 1502, 1460, 1200, 1095, 1028, 970 cm−1; UV (MeOH) λmax (log ε) 225 (0.706), 284 (0.182) nm; CD (MeOH, Δε) λmax 341 (+ 9.90), 307 (− 2.53), 274 (+ 13.4), 242 (− 41.6), 215 (+ 42.1) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 341.0996 [M + Na]+ (calc. For C17H18O6 + Na, 341.1001). 2.3.2. Astragaisoflavan B (2) Colorless needle crystal; [α]20 D –11 (c 0.5, MeOH); IR (neat) νmax 3380, 2925, 1660, 1600, 1500, 1460, 1190, 1090 cm− 1; UV (MeOH) λmax (log ε) 225 (2.235), 301 (0.691) nm; CD (MeOH, Δε) λmax 337 (−10.1), 245 (+45.6) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 319.1176 [M + H]+ (calc. for C17H18O6 + H, 319.1182).

3. Results and discussion Astragaisoflavan A (1) was obtained as optically active and colorless crystals, with the molecular formula of C17H18O6 determined by HRESIMS (m/z 341.0996 [M + Na]+). A hydroxyl peak was observed at 3467 cm−1 in IR spectrum and the presence of an α,β-unsaturated ketone carbonyl group was indicated by the absorption at 1665 cm−1 and 13C NMR peak at δC 191.0. The 13C NMR (DEPT) spectrum (Table 1) exhibited 17 carbon signals including two methoxys, two methylenes, six methines, and seven quaternary carbons. The 1H NMR spectrum (Table 1) gave a characteristic proton signal of the isoflavan nucleus at δH (4.63 m, 3.84 t; 4.10 m; 2.23 m, 2.02 dd) and an ortho

2.3.3. Astragaisoflavan C (3) Colorless needle crystal; [α]20 D + 70 (c 0.7, MeOH); IR (neat) νmax 3400, 2850, 1710, 1610, 1506, 1465, 1093, 1039 cm− 1; UV (MeOH) λmax (log ε) 224 (2.720), 274 (0.481) nm; CD (MeOH, Δε) λmax 285 (+ 1.98), 266 (− 0.80), 223 (− 4.19) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 375.1414 [M + Na]+ (calc. for C18H24O7 + Na, 375.1420). Table 1 1 H NMR (400 MHz) and 13C NMR (100 MHz) data of compounds 1, 2, 3 and 5 (CD3OD). 1

2

3

5

No.

δH (J in Hz)

δC

δH (J in Hz)

δC

δH (J in Hz)

δC

δH (J in Hz)

δC

2a 2b 3 4a 4b 5a 5b 6a 6b 7 8a 8b 9 10 1ʹ 2ʹ 3ʹ 4ʹ 5ʹ 6ʹ 3ʹ-OMe 4ʹ-OMe 9-OMe 2ʹ-OHa 10-OHa

4.63 m 3.84 t (9.6) 4.10 m 2.23 m 2.02 dd (13.6, 11.2) 6.72 d (9.6)

74.9 t

70.6 t

6.06 dd (10.0, 1.6)

127.7 d

5.98 dd (10.0, 2.0)

126.3 d

4.29 m 3.68 m 3.91 m 2.02 m 1.98 m 1.92 m 1.90 m 2.67 m 2.15 d (16.4)

73.0 t

31.7 d 38.3 t

3.78 m 3.54 dd (11.6, 11.2) 3.89 m 2.68 dd (14.4, 1.2) 2.35 t (12.8) 2.08 td (12.8, 5.6) 1.80 m 2.78 m 2.25 m

65.6 t

150.1 d

4.83 m 4.13 dd (9.6, 4.4) 3.60 m 2.25 dd (14.4, 8.0) 2.13 dd (14.8, 10.4) 6.71 d (10.0)

5.62 d (2.0)

191.0 s 108.9 d

5.59 d (2.0)

190.4 s 104.9 d

6.45 d (8.8) 6.79 d (8.8) 3.77 s 3.79 s

177.2 s 66.1 s 121.2 s 149.9 s 137.8 s 153.3 s 104.6 d 123.3 d 61.2 q 56.4 q

6.53 d (8.4) 6.98 d (8.8) 3.77 s 3.81 s

176.7 s 67.4 s 121.2 s 149.6 s 137.5 s 153.4 s 104.8 d 123.8 d 61.0 q 56.3 q

a

8.99 s 6.02 s

Recorded in DMSO-d6.

32.1 d 38.5 t

8.96 s 6.02 s

148.8 d

3.09 d (14.4) 2.68 dd (14.4, 1.2)

6.54 d (8.8) 6.86 d (8.8) 3.84 s 3.88 s 3.27 s 8.86 s 5.08 s

31.6 d 36.8 t 34.6 t 37.8 t 211.1 s 44.5 t 102.1 s 71.1 s 122.1 s 149.8 s 137.5 s 153.0 s 104.3 d 123.0 d 61.0 q 56.2 q 47.9 q

29.5 d 39.3 t 36.4 t 33.3 t

5.24 s

199.0 s 108.1 d

6.47 d (8.4) 6.80 d (8.8) 3.67 s 3.75 s

175.4 s 65.7 s 120.3 s 148.8 s 136.6 s 152.2 s 103.7 d 122.1 d 60.7 q 56.1 q

8.97 s 5.59 s

K. Guo et al. / Fitoterapia 114 (2016) 34–39

37

Table 2 1 H NMR (400 MHz) and 13C NMR (100 MHz) data of compounds 4 and 12 (CD3OD). 4

12

no.

δH (J in Hz)

δC

HMBC (H → C)

No.

δH (J in Hz)

δC

2a 2b 3 4a 4b 5 6 7 8 9 10 1ʹ 2ʹ 3ʹ 4ʹ 5ʹ 6ʹ 2ʺa 2ʺb 3ʺ 4ʺa 4ʺb 5ʺ 6ʺ 7ʺ 8ʺ 9ʺ 10ʺ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 2ʹ-OMe 5ʹ-OMe 4‴-OMe 5‴-OMe

4.10 dd (10.4, 2.8) 4.00 m 3.46 m 2.88 dd (16.0, 6.0) 2.56 dd (16.0, 5.6) 6.64 d (8.4) 6.18 dd (8.4, 2.4)

70.1 t

C-4, 9, 1ʹ C-2, 4, 10, 1ʹ, 2ʹ, 6ʹ C-2, 3, 5, 9, 10, 1ʹ

130.9 d 109.4 d 157.4 s 103.9 d 156.1 s 113.6 s 124.7 s 142.9 s 143.9 s 146.6 s 142.5 s 109.7 d 71.1 t

C-4, 7, 8, 9 C-7, 8, 10

4.34 ddd (10.4, 3.2, 1.6) 4.07 t (10.0) 3.53 m 3.00 dd (15.6, 10.4) 2.91 ddd (16.0, 5.6, 1.2) 6.94 d (8.0) 6.38 dd (8.0, 2.4)

69.7 t

32.4 d 30.4 t

2a 2b 3 4a 4b 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 3′-OMe 4′-OMe

33.2 d 32.2 t

C-2ʺ, 4ʺ, 10ʺ, 1‴, 2‴, 6‴ C-2ʺ, 3ʺ, 5ʺ, 9ʺ, 10ʺ, 1 ‴

131.2 d 109.1 d 157.6 s 103.8 d 156.2 s 114.6 s 129.0 s 147.7 s 110.6 d 154.0 s 141.9 s 123.1 d 60.9 q 61.6 q 56.4 q 61.4 q

C-4ʺ, 7ʺ, 8ʺ, 9ʺ C-7ʺ, 8ʺ, 10ʺ

6.00 d (2.4)

5.99 s 4.00 m 3.84 m 3.17 m 2.77 dd (15.6, 10.8) 2.61 dd (16.0, 4.4) 6.80 d (8.4) 6.28 dd (8.4, 2.1) 6.17 d (2.4)

6.82 s

6.83 s 3.45 s 3.85 s 3.84 s 3.90 s

C-6, 7, 9, 10

C-3, 1ʹ, 2ʹ, 4ʹ, 5ʹ-OMe C-4ʺ, 9ʺ, 1‴

O H

C‴, 5‴, 6‴

C-3ʺ, 1 ‴, 2 ‴, 4 ‴ C-2ʹ C-5ʹ C-4‴ C-5 ‴

and C(5′)H-C(6′)H (Fig. 2). The inspection of HMBC spectrum allowed for a complete assignment of all signals in this structure based on the cross peaks of H-2a with C-9, C-1′; of H-3 with C-10, C-1′, C-2′, C-6′; of H-4a with C-5, C-9, C-1′; of H-5 with C-4, C-7, C-9; of H-8 with C-6, C10; of H-5′ with C-1′, C-3′; of H-6′ with C-3, C-2′, C-4′ (Fig. 2). Thus, the planar structure of compound 1 was established. In the NOESY experiment, H-3 showed corrections with H-2a, H-4a, OH-10, OH-2′ and H-6′, which indicated both the location and the α-

O

O

OH

H

OCH3

OH

O

OH

OCH3

H

H3CO H3CO

O

OCH3

3

O

H

H

OCH3

H3CO

OH

COSY

OH OH

4 1H-1H

OH OCH3

OH 2

O

OCH3 O

OCH3

OH 1

6.44 d (8.8) 6.78 d (8.8) 3.85 s 3.92 s

130.4 d 107.9 d 155.3 s 103.2 d 154.8 s 114.7 s 120.3 s 147.5 s 135.4 s 151.1 s 103.7 d 121.8 d 55.8 q 61.0 q

C-6ʺ, 7ʺ, 9ʺ, 10ʺ

aromatic proton spin system (δH 6.79 d; 6.45 d). The NMR data of 1 were in complete agreement with those of 5 (Table 1) except the only difference that two methylene signals (δH 1.90, 1.92; 2.15, 2.67; δC 36.4; 33.3) in 5 were replaced by an olefinic bond signals (δH 6.72; 6.06; δC 150.1; 127.7) in 1 at C-5 and C-6, which could be further confirmed by a detailed analysis of the 2D NMR (1H − 1H COSY, HMQC and HMBC) spectra. The 1H\\1H COSY spectrum of compound 1 displayed three isolated spin-coupling systems: C(2)H2-C(3)H-C(4)H2; C(5)H-C(6)H-C(8)H;

O

6.35 d (2.4)

32.2 d 30.2 t

HMBC

Key 1H-1H COSY and HMBC correlations of compounds 1–4 Fig. 2. Key 1H\ \1H COSY and HMBC correlations of compounds 1–4.

OCH3

38

K. Guo et al. / Fitoterapia 114 (2016) 34–39

orientation of the hydroxyl at C-10 (assuming H-3 to be α-oriented). The CD spectrum (Fig. 3) of compound 1 displayed a strong positive Cotton effect at the 1Lb transition region (260–300 nm), which indicated the 3R absolute configuration of 1 [15]. Such an allocation was strongly supported by the same Cotton effect at the 1Lb transition region in the CD spectrum of (3R)-isomucronulatol (12) [1]. Accordingly, the (3R,10R) absolute configuration of 1 could be assigned and the structure of astragaisoflavan A (1) was established as (3R,10R)-10-hydroxy-5,8dien-7-carbonylisomucronulatol. Astragaisoflavan B (2) was obtained as optically active and colorless crystals, with the same molecular formula as 1 of C17H18O6 determined by HRESIMS (m/z 319.1176 [M + H]+). The IR and UV spectra of 2 were nearly the same as 1, while the orientation of specific rotation was reverse. Although the 1H and 13C NMR data of compound 2 and 1 were very similar, some obvious differences were found between the chemical shifts of both proton and carbon signals at C-2, C-3 and C-4 (Table 1). The NOESY experiment of 2 gave the correlations: H-3 with H-2b, H-4b and H-6′; OH-10 with H-2a and H-4a. Thus, assuming H-3 to be β-oriented, H-2b and H-4b were β-orientation, while H-2a, H-4a and OH-10 were α-orientation. The CD spectrum (Fig. 3) of compound 2 displayed a negative Cotton effect at the 1Lb transition region (260–300 nm), indicating the rare 3S absolute configuration of 2 [15], which was further confirmed with comparison to that of 1. Therefore, astragaisoflavan B (2) was assigned with the (3S,10R) absolute configuration and established as (3S,10R)-10-hydroxy-5,8-dien-7-carbonylisomucronulatol. Astragaisoflavan C (3) was obtained as optically active and colorless crystals, with the molecular formula of C18H24O7 determined by HRESIMS (m/z 375.1414 [M + Na]+). The IR spectrum suggested the presence of hydroxyl (3400 cm−1) and carbonyl (1710 cm−1) groups. Comparing the NMR data of 3 with that of 5 revealed the similar carbon framework between 3 and 5, except for some differences in ring A. The olefinic bond signals (δH 5.24; δC 108.1; 175.4) in 5 at C-8 and C-9 were replaced by a saturated moiety (δH 3.09, 2.68; δC 44.5; 102.1) in 3, and a new methoxy group occurred at C-9 with the NMR data (δH 3.27; δC 47.9) which was verified by the HMBC correlation from OMe-9 to C-9 (Fig. 2). The NOESY correlations of H-3 with H-2a, H-6′ and OH-10, of OH-10 with H-3 and H-8a, and of OMe-9 with H-3 and OH-10 revealed a co-facial relationship of H-3, OH-10 and OMe-9. Assuming H-3 to be α-oriented, OH-10 and OMe-9 both were α-orientation. The CD spectrum of compound 3 (Fig. 3) showed a positive Cotton effect and matched very well with that of (3R)-isomucronulatol at the 1Lb transition region (260–300 nm). Consequently, the (3R,9R,10S) absolute configuration was assigned and the structure of astragaisoflavan C (3) was

established as (3R,9R,10S)-10-hydroxy-9-methoxy-7-carbonyl-5,6,8trihydroisomucronulatol. Astragaisoflavan D (4) was obtained as optically active and amorphous yellow solids, with the molecular formula of C34H34O10 determined by HRESIMS (m/z 625.2044 [M + Na]+). IR spectrum suggested the existence of hydroxyl (3400 cm−1) and aromatic ring (1597, 1502, and 1458 cm− 1) absorptions. The 13C NMR data of 4 (Table 2) gave 30 skeletal carbon signals including four methylenes with two linked to an oxygen atom, eleven methines and fifteen quaternary carbons, together with four substituted methoxy carbon signals. The 1H NMR data of 4 (Table 2) displayed two typical sets of the isoflavan nucleus signals at δH (4.10 dd, 4.00 m; 3.46 m; 2.88 dd, 2.56 dd) and δH (4.00 m, 3.84 m; 3.17 m; 2.77 dd, 2.61 dd), respectively. Two ABX aromatic proton spin system (δH 6.64 d, 6.18 dd, 6.00 d; 6.80 d, 6.28 dd, 6.17 d) and three single aromatic proton (δH 5.99, 6.82, 6.83) were further observed in the 1H NMR spectrum. These signals and the molecular mass of 602 both indicated that compound 4 could be a dimeric isoflavan. Four isolated spin-coupling systems: C(2)H2-C(3)H-C(4)H2, C(5)HC(6)H, C(2″)H2-C(3″)H-C(4″)H2 and C(5″)H-C(6″)H, were identified from the 1H\\1H COSY spectrum (Fig. 2). In the HMBC spectrum (Fig. 2), the cross peaks of H-3 with C-1′, C-2′, C-6′; of H-6′ with C-3, C-2′, C-4′, OMe-5′; of OMe-2′ with C-2′; of OMe-5′ with C-5′ revealed a partial structure in the ring B of one isoflavan unit and the respective location of two hydroxyls at C-3′ and C-4′, while the partial structure in the ring B of another isoflavan unit and a hydroxyl located at C-2‴ were also deduced by analysis of the HMBC spectrum as follows: the cross peaks of H-3″ with C-1‴, C-2‴, C-6‴; of H-3‴ with C-1‴, C-5‴; of H-6‴ with C3″, C-2‴, C-4‴; of OMe-4‴ with C-4‴; of OMe-5‴ with C-5‴. Connectivity of the two isoflavan units in compound 4 could be deduced by the HMBC cross peaks (Fig. 2) from H-6 to C-7/C-8/C-10, H-8 to C-6/C-7/ C-9/C-10, H-6″ to C-7″/C-8″/C-10″ and H-8″ to C-6″/C-7″/C-9″/C-10″, which was further corroborated by the observation of NOESY correlations between H-6 and H-6″. Finally the existence of an ether linkage between C-7 and C-7″ was ascertained by calculating the molecular mass of compound 4. Besides, the resonance at C-7 and C-7″ of compound 4 both were shifted downfield by about 2 ppm compared with the corresponding carbon resonance at C-7 of (3R)-isomucronulatol (12) (Table 2), which showed the interisoflavan ether linkage between C-7 and C7″ in 4. The CD data of compound 4 (Fig. 3) exhibited a strong positive Cotton effect at the 1Lb transition region (260–300 nm), indicating the (3R,3ʺR) absolute configuration of 4, which was strongly supported by the same Cotton effect of (3R)-isomucronulatol [1]. Thus, the structure

Fig. 3. CD spectra of compounds 1–4.

K. Guo et al. / Fitoterapia 114 (2016) 34–39

of astragaisoflavan D (4) was characterized as (3R,3ʺR)-3′,4′,2‴-trihydroxy-2′,5′,4‴,5‴-tetramethoxy-(7–7″)-oxy-biisoflavan. By comparing the spectroscopic data with that reported in the literatures, thirteen known flavonoids were identified as oxytropisoflavan B (5) [1], rhamnocitrin (6) [16], rhamnocitrin-3-O-β-D-glucopyranoside (7) [17], 7-hydroxy-4′-methoxyisoflavone (8) [18], calycosin (9) [19], pratensein (10) [20], (3R)-8,2′-dihydroxy-7,4′- dimethoxyisoflavane (11) [21], (3R)-isomucronulatol (12) [22], (3R)-mucronulatol (13) [23], isoliquiritigenin (14) [24], homobutein (15) [25], (3R)-violanone (16) [26], and liquiritigenin (17) [27]. Compound 1, 2, 3 and 5 all possessed an isoflavan skeleton with a ketone carbonyl group in ring A, which was rarely found in natural products. Compound 4 was a new biisoflavan consisting of two isoflavan units linked through an interesting C-O-C bond, as most dimeric flavonoids have the C-dimeric structures (for instance, ginkgetin). Compound 5 was first identified from genus Astragalus and all of the known compounds above (6–17) were isolated from A. hoantchy for the first time. Among the above flavonoids, compound 1–4 and 11–13 belong to isoflavanes, 6 and 7 are flavones, 8–10 belong to isoflavones, 14 and 15 are chalcones, 16 belongs to isoflavanone, and 17 is a flavanone. Compound 1 and 4 were selected to evaluate their antifungal effect against A. solani in vitro by measuring the radial growth of mycelia. Compound 1 did not show obvious inhibitory activity at the testing concentration of 100 μg/mL, while 4 exhibited a moderate inhibitory activity with the IC50 value of 173.3 μg/mL. Conflict of interest The authors declared no conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31570354, 21302195, and 31300290), Agricultural Biotechnology Research and Development Program of Gansu Province (GNSW-2015-25), Research and Development Project of New Technology and Equipment of Special Program in the Field of Agriculture Ecological Environment Projection of Gansu Province, Cooperation Program to Gansu Province of Lanzhou Branch of the Chinese Academy of Sciences, and 135 Major Breakthrough Project of the Chinese Academy of Sciences. Appendix A. Supplementary data 1D NMR, 2D NMR, IR spectrum and HRESIMS of compounds 1–4. Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.fitote.2016.08.009. References [1] W.C. Chen, R. Wang, Y.P. Shi, Flavonoids in the poisonous plant Oxytropis falcate, J. Nat. Prod. 73 (2010) 1398–1403.

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