Original Article

259

Two New Flavonoids and Biological Activity of Astragalus abyssinicus (Hochst.) Steud. ex A. Rich. Aerial Parts

Authors

R. A. El Dib1, 2, H. S. M. Soliman2, 3, M. H. Hussein4, H. G. Attia5

Affiliations

1



Key words ▶ Astragalus abyssinicus ● ▶ Fabaceae ● ▶ Isolation ● ▶ Flavonoids ● ▶ Antibacterial activity ● ▶ Antioxidant activity ●

 Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh Saudi Arabia  Department of Pharmacognosy, Faculty of Pharmacy, Helwan University, Cairo, Egypt 3  Department of Pharmacognosy, College of Pharmacy, King Khalid University, Abha, Kingdom of Saudi Arabia 4  Department of Pharmacognosy, College of Pharmacy, Al-Azhar University, Cairo, Egypt 5  Department of Pharmacognosy, College of Pharmacy, Najran Univeristy, Najran, Kingdom of Saudi Arabia 2

Abstract



2 new flavonoid glycosides, kaempferol 3-O(4″,6″-di-O-α-L-rhamnopyranosyl)-β-Dglucopyranoside (1) and quercetin 3-O-(4″,6″-diO-α-L-rhamnopyranosyl)-β-D-glucopyranoside (2), were isolated from the n-butanol soluble fraction of the methanol extract (BF) of Astragalus abyssinicus aerial parts, together with 3 known compounds, rutin (3), kaempferol

Introduction

▼ received 28.02.2014 accepted 05.05.2014 Bibliography DOI  http://dx.doi.org/ 10.1055/s-0034-1377003 Published online: June 18, 2014 Drug Res 2015; 65: 259–265 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence Dr. R. A. El Dib, Associate Professor Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia Department of Pharmacognosy Faculty of Pharmacy Helwan University Cairo Egypt Tel.:  + 966/1180/52 820 Tel.:  + 20/2/25541 601 Fax:  + 20/2/25541 601 [email protected]

Astragalus (Hochst.) Steud. ex A. Rich. is generally considered the largest genus of family Fabaceae and one of the largest genera of vascular plants on earth, comprising about 2 000 species distributed throughout the temperate regions of the world, located in Europe, Asia, North America and tropical African mountains [1, 2]. The word Astragalus was derived from 2 Greek words “Astron” meaning star and “Gala” meaning milk, for the belief that the presence of Astragalus plants in grassland will increase the milk yield of livestock [3]. Most Astragalus species are sunloving and prefer a light, porous soil, many are drought-resistant [1]. A. abyssinicus is an endemic endangered plant [4]. It is a perennial spineless plant, shrubby herb, up to 1.5 m with erect and glabrous, very thick stem. The leaves are sessile with elliptic, oblong, glaucous green leaflets. The plant’s flowers are red or purple, 6 mm long ageing blue and present in long racemes [4, 5]. The fruits are legumes, linear 2.5 cm long, divided inside by longitudinal membrane [5]. Astragalus species are known to be rich in cycloartane-type triterpene glycosides [6] and their roots to be rich in polysaccharides and saponins [7–10]. In addition, several flavonoids and isoflavonoids have been isolated from different Astragalus species [11–15].

3-O-β-D-rutinoside (4) and 5,7,4′-trihydroxy3′-methoxyisoflavone (5). The structures of the isolated compounds were characterized on the basis of UV, NMR and negative ESI-MS analyses. The BF fraction showed in vitro weak antibacterial activity against Staphylococcus aureus, while 2 and 3 exhibited in vitro antioxidant activity higher than ascorbic acid using DPPH free radical scavenging activity method.

Some Astragalus species have been reported as having immunostimulant, cardiovascular and antiviral activities [3, 7]. Moreover, the roots of various Astragalus spp. are used as antiperspirants, diuretics, tonic agents and are used for treatment of diabetes mellitus, nephritis, leukaemia and uterine cancer. The hepatoprotective, antioxidative, immunostimulant and antiviral properties of Astragalus species have been examined [16] and their anti-inflammatory, analgesic, hypotensive, sedative and cardiotonic activities are also reported [17]. In addition, antibacterial activity is also ascribed to some Astragalus spp., including A. siculus [18], A. gummifer [19] and A. melanophrurius [20]. This paper reports the first phytochemical and biological investigation of A. abyssinicus aerial parts, comprising the isolation and identification of 2 new flavonoid glycosides (1,2), along with 3 known flavonoids (3–5) from the n-butanol soluble portion of the methanol extract (BF).

Experimental



General

NMR spectra were recorded on JEOL 500 and Bruker advanced DRX-500 spectrometers operating at 500 MHz and 125 MHz for 1H and 13C NMR, respectively. Few 1H NMR spectra were recorded on JEOL GX270 NMR spectrometer. The

El Dib RA et al. Flavonoids from Astragalus abyssinicus  …  Drug Res 2015; 65: 259–265

Downloaded by: Chinese University of Hong Kong. Copyrighted material.



260 Original Article

Plant material

The aerial parts of A. abyssinicus were collected from Abha city, Aseer region, Saudi Arabia in May 2005. The identity of the plant was confirmed by Prof. Hussein Elwadei, Department of Plant Taxonomy, College of Science, King Khalid University, Saudi Arabia. The plant was air dried in shade then powdered. Voucher specimens are kept in the Department of Pharmacognosy, College of Pharmacy, King Khalid University, Abha, Saudi Arabia.

Extraction and isolation

The powder of air-dried aerial parts of A. abyssinicus (3.9 kg) was extracted with CHCl3 and the marc left was subjected to extraction with 70 % MeOH under reflux (3 × 6 L). The MeOH extract was dried under reduced pressure and the residue (180 g) was suspended in 500 ml dist. H2O and fractionated with n-BuOH saturated with H2O (3 × 500 ml). The resulting n-BuOH fraction (BF) was then evaporated under reduced pressure to yield a 60 g residue, which was chromatographed over a polyamide column (5 × 100 cm) eluted with H2O and stepwise gradient with H2O/MeOH mixtures up to 70% MeOH. The individual fractions (400 ml each) were collected into 22 fractions (A-V) by TLC screening. Based on their TLC profile, fractions were selected for further column chromatography to afford pure compounds. Fraction B (7.5 g, 100 % H2O) was chromatographed over silica gel column using CHCl3/MeOH eluent (90:10 to 60:40). The fractions collected (120 ml each) were combined to afford 18 fractions (1–18) by the aid of TLC. Fraction 12 (1 g, 70% CHCl3/ MeOH) was applied over RP C18 column using H2O/MeOH mixtures with decreasing polarity to yield 1 (26 mg, 75:25 H2O:MeOH) and 2 (211 mg, 80:20 H2O:MeOH). Fraction N (715 mg, 60 % H2O) was chromatographed over silica gel column using 90% CHCl3/MeOH to afford 8 fractions (1–8). Fractions 2 (52 mg) and 3 (41 mg) were individually subjected to preparative TLC using solvent system S5 to yield 3 (22 mg) and 4 (13 mg), respectively. Fraction U (53 mg, 30 % H2O) was subjected to preparative TLC with S2 for elution to yield 5 (6 mg).

Kaempferol 3-O-(4″,6″-di-O-α-L-rhamnopyranosyl)-βD-glucopyranoside (1)

Yellow amorphous powder; Chromatographic properties: Rfvalues 0.52 (S4), 0.62 (S3), dark purple spot under long UV-light (UV-365 nm) turned to greenish yellow fluorescence and green colour with Naturstoff and FeCl3 spray reagents, respectively; El Dib RA et al. Flavonoids from Astragalus abyssinicus …  Drug Res 2015; 65: 259–265

UV/vis λ max: (MeOH) 266, 346; ( + NaOMe) 274, 412; ( + NaOAc) 274, 363; ( + NaOAc/H3BO3) 267, 348; ( + AlCl3) 271, 361; ( + AlCl3/ HCl) 274, 346.1H NMR (500 MHz, DMSO-d6) and 13C NMR (125  ▶  Table 1, 2, respectively. Negative MHz, DMSO-d6): data see ● ESI-MS/MS: m/z 738.91 [M-H]-, 593.06 [M-deoxyrhamnosyl]-, 447.01 [M-deoxydirhamnosyl]-, 285.74 [M-deoxydirhamnosylglucosyl]- = [kaempferol-H]-.

Quercetin 3-O-(4″,6″-di-O-α-L-rhamnopyranosyl)-β-Dglucopyranoside (2)

Dark brown amorphous powder; Chromatographic properties: Rfvalues 0.32 (S4), 0.44 (S3), dark purple spot under long UV-light (365 nm) turned to orange fluorescence and green colour with Naturstoff and FeCl3 spray reagents, respectively; UV/vis λ max: (MeOH) 260, 357; ( + NaOMe) 272, 410; ( + NaOAc) 270, 374; ( + NaOAc/H3BO3) 264, 379; ( + AlCl3) 271, 432; ( + AlCl3/HCl) 258, 352.1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz,  ▶  Table 1, 2, respectively. Negative ESI-MS/ DMSO-d6): data see ● MS: m/z 755.05 [M-H]-, 609.25 [M-deoxyrhamnosyl]-, 463.96 [M-deoxydirhamnosyl]-, 301.06 [M-deoxydirhamnosylglucosyl]- = [quercetin-H]-.

Rutin (3)

Yellow amorphous powder; Chromatographic properties: Rfvalues 0.45 (S4), 0.40 (S6), dark purple spot under long UV-light (365 nm) turned to orange fluorescence and green colour with Naturstoff and FeCl3 spray reagents, respectively; UV/vis λ max: (MeOH) 257, 357; ( + NaOMe) 272, 411; ( + NaOAc) 273, 396; ( + NaOAc/H3BO3) 261, 378; ( + AlCl3) 274, 436; ( + AlCl3/HCl) 269, 400. 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz,  ▶  Table 1, 2. Negative HRESI-MS/MS: m/z DMSO-d6): data see ● 609.34 [M-H]-, 463.73 [M-deoxyrhamnosyl]-, 301.03 [M-deoxyrhamnosylglucosyl]- = [quercetin-H]-.

Kaempferol 3-O-β-D-rutinoside (4)

Yellow amorphous powder; Chromatographic properties: Rfvalues 0.52 (S4), 0.46 (S6), dark purple spot under long UV-light (365 nm) turned to greenish yellow fluorescence and green colour with Naturstoff and FeCl3 spray reagents, respectively; UV/ vis λ max: (MeOH) 266, 351; ( + NaOMe) 274, 402; ( + NaOAc) 274, 389; ( + NaOAc/H3BO3) 267, 352; ( + AlCl3) 271, 351; ( + AlCl3/HCl)  ▶  Table 1. 274, 346.1H NMR (500 MHz, DMSO-d6): data see ●

5,7,4′-trihydroxy-3′-methoxyisoflavone (5)

Yellow amorphous powder; Chromatographic properties: Rfvalues 0.41 (S2), 0.47 (S1), dark purple spot under long UV-light (365 nm) turned to yellow fluorescence and green colour with Naturstoff and FeCl3 spray reagents, respectively. 1H NMR (270  ▶  Table 1. MHz, CD3OD): data see ●

Acid hydrolysis

For determination of the aglycones and the sugar moieties, complete acid hydrolysis was carried out for compounds 1–4 by refluxing 4–5 mg of each compound with 1.5 N HCl in aq. MeOH (1:1) for 2 h. The product was then extracted with CHCl3 and the extract was subjected to CoTLC investigation alongside with the authentic aglycones. The mother liquor was neutralized with BaCO3 and used for study of the sugar moieties using CoTLC with authentic sugars [21].

Downloaded by: Chinese University of Hong Kong. Copyrighted material.

δ-values are reported in ppm relative to TMS in the convenient deuterated solvents, while coupling constants (J) are reported in Hz. ESI-MS analyses were recorded on a Thermo-LC mass spectrometer. UV analyses of pure samples were recorded in MeOH and separately with different diagnostic shift reagents on a Shimadzu UV-2550 spectrophotometer. Column chromatography was carried out on various adsorbents including polyamide S (Fluka, Steinheim, Switzerland) and silica gel 230–400 mesh and silica gel RP C18 (E. Merck, Darmstadt, Germany). TLC was performed on pre-coated silica gel F254 plates (E. Merck, Darmstadt, Germany). Solvent systems: S1: [CHCl3/MeOH (9:1 v/v)], S2: [CHCl3/MeOH (9:0.5 v/v)], S3: [CHCl3/MeOH/H2O (9:6:1 v/v)], S4: [CHCl3/MeOH/H2O (9:4:0.5 v/v)], S5: [CHCl3:MeOH:H2O (9:3.5: 0.2)] and S6 [CHCl3/MeOH/H2O (9:3:0.5 v/v)] were used for TLC. The detection of spots was done in UV-light (254 and/or 365 nm) and spraying reagents e. g. Naturstoff reagent (a) 1% diphenyl boryloxyethanolamine in ethanol, (b) 5% polyethylene glycol 400 in MeOH or by spraying with FeCl3 (1% in EtOH).

Original Article

1a

Aglycone moieties 2 3 4 5 12.58 s 6 6.11 brs 7 8 6.31 brs 1' 2' 7.90 d (8.4) 3' 6.84 d (8.4) 4' 5' 6.84 d (8.4) 6' 7.90 d (8.4) -OCH3-3' Sugar moieties 1'' 5.45 d (6.9) 2'' 3'' 3.03–3.84 m, 4'' remaining sugar 5'' protons 6'' 1''' 5.01 brs 2''' 3.03–3.84 m, 3''' remaining sugar 4''' protons 5''' 6''' 0.92 d (6.1) 1'''' 4.28 brs 2'''' 3.03–3.84 m, 3'''' remaining sugar 4'''' protons 5'''' 6'''' 0.76 d (6.1)

2b

3c

4d

5e 8.12 s

12.61 s 5.95 brs

12.54 s 6.14 brs

12.51 s 6.13 brs

6.22 d (1.8)

6.13 brs

6.34 brs

6.34 brs

6.34 d (1.8)

7.40 brs

7.48 m

7.93 d (8.4) 6.83 d (8.4)

7.18 d (2.1)

6.74 d (8.4) 7.47 d (8.4)

6.79 d (8.4) 7.48 m

6.83 d (8.4) 7.93 d (8.4)

6.86 d (8.1) 6.99 dd (8.1, 2.1) 3.90 s

5.47 d (7.7)

5.29 d (6.1)

5.25 d (6.9)

2.95–3.62 m, remaining sugar protons

2.95–3.52 m, remaining sugar protons

5.00 brs 2.95–3.62 m, remaining sugar protons

4.35 brs 2.95–3.52 m, remaining sugar protons

4.33 brs

0.94 d (6.1) 4.30 brs

0.94 d (6.1)

0.93 (d, 6.1)

Table 1  1H NMR data of compounds 1–5 (δH in ppm, J in parentheses in Hz).

2.95–3.62 m, remaining sugar protons 0.77 d (6.1)

a–d

 500 MHz in DMSO-d6

e

 270 MHz in MeOH-d4

Antibacterial activity Material

Mueller-Hinton Agar (Oxoid) was used as medium for Antimicrobial Susceptibility Testing (AST). The used formula was composed of Beef dehydrated infusion (300 g), casein hydrolysate (17.5 g), starch (1.5 g) and agar (17 g). The pH was adjusted as follows 7.3 ± 0.1 at 25 °C. to 1 L of dist. H2O, 38 g of medium were added, followed by boiling the suspension to dissolve the medium completely. The medium was dispensed in sterile Petri dishes after sterilization by autoclaving at 121 °C for 20 min. The solutions were tested for their antibacterial activity against the bacterial strains Staphylococcus aureus, Escherichia coli and Klebsiella pneumonia, which were obtained from the Department of Microbiology, College of Medicine, Nejran University. The CHCl3 and the MeOH extracts, as well as extracts resulting from fractionation of the latter, including H2O and n-BuOH saturated with H2O (BF) extracts, were used for the study. In addition, compounds 2 and 3 were tested for their antibacterial activity against the bacterial isolates at concentration of 10 mg/dl.

Methods

Filter paper discs were prepared and sterilized before use in the autoclave at 121 °C for 20 min. 3 filter paper discs were soaked in each of the tested solutions for 20 min. The filter paper discs were left to air dry for 30 min in sterile plates. The bacterial

strains were inoculated on Mueller-Hinton agar plates as recommended by NCCLS (2004) as follows: 4–5 isolated colonies of similar morphology from each bacterial isolate were taken by a loop, inoculated into 5 ml sterile nutrient broth and incubated for 2–5 h at 35 °C until turbidity was obtained. This turbidity was then adjusted with saline to 0.5 McFarland Standard (prepared by mixing 0.05 ml of 1.175 % BaCl2.H2O with 9.95 ml of 1 % H2SO4). This equals a cell density of 108 cells/ml approximately. From these broth suspensions, inocula were applied by sterile swabs on Muller-Hinton agar. Inside the laboratory safety cabinet, sterile forceps were used to pick up the dried paper discs to be applied to the center of plates containing the bacterial isolates. Plates were incubated at 37 °C for 24 h. After incubation, each plate was examined for the presence or absence of inhibition zone around the paper disc. Regardless of zone size any discernible growth within the zone of inhibition is indicative of resistance.

Antioxidant activity Materials

DPPH (1,1-Diphenyl-2-picrylhydrazyl) free radicals and ascorbic acid (Sigma-Aldrich Chemicals Co., USA) were used. DPPH was prepared in absolute EtOH at a concentration of 0.1 mM, while ascorbic acid was prepared as serial dilutions in the range of 0–25 µg/ml in dist. H2O for plotting the calibration curve of the

El Dib RA et al. Flavonoids from Astragalus abyssinicus  …  Drug Res 2015; 65: 259–265

Downloaded by: Chinese University of Hong Kong. Copyrighted material.

Position

261

Table 2 

13

C NMR data of compounds 1–3 (125 MHz, DMSO-d6).

Position Aglycone moieties 2 3 4 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' Sugar moieties 1'' 2'' 3'' 4'' 5'' 6'' 1''' 2''' 3''' 4''' 5''' 6''' 1'''' 2'''' 3'''' 4'''' 5'''' 6''''

1

2

3

δC

δC

δC

157.6 133.1 177.8 161.7 99.2 165.1 94.6 157.2 104.5 121.7 131.4 115.8 160.3 115.8 131.4

156.8 132.7 177.0 157.9 100.0 161.5 99.1 154.4 101.3 122.0 116.1 145.5 149.8 115.6 121.7

157.0 133.7 177.8 161.7

99.6 74.7 77.5 78.3 77.0 69.0 101.3 71.2 70.9 72.4 68.9 17.7 101.4 71.2 70.9 72.4 68.9 18.1

100.4 72.3 77.6 77.7 76.3 68.8 101.0 71.0 70.8 71.1 70.8 17.7 101.1 71.0 70.8 71.1 70.8 18.3

99.3 74.6 76.9 70.9 76.3 68.7 101.2 71.0 71.0 72.3 70.4 18.2

165.0

104.3 121.6 116.7 145.3 149.0 115.7 122.1

reference drug. The antioxidant activity was assessed on the basis of the scavenging activity of the stable 1,1-diphenyl-2picrylhydrazyl (DPPH) free radicals [22]. Tested extracts/compounds included MeOH and BF extracts as well as compounds 2 and 3.

Methods

In a flat bottom 96 well-microplate, a total test volume of 200 µl was used. In each well, 20 µl of different concentrations (0–40 µg/ ml final concentration) of tested sample were mixed with 180 µl of ethanolic DPPH and incubated for 30 min at 37ºC. Then photometric determination of absorbance at 515 nm was performed by microplate ELISA reader. Triplicate wells were prepared for each concentration and the average was calculated.

Results and Discussion



The structures of the isolated compounds were elucidated on the basis of UV, 1H and 13C NMR and negative ESI-MS analyses and by comparison of their data with published data of structurally related compounds [23–25]. On the basis of its chromatographic properties and UV spectral data, 1 was expected to be a flavonol 3-O-glycoside [25]. ComEl Dib RA et al. Flavonoids from Astragalus abyssinicus …  Drug Res 2015; 65: 259–265

plete acid hydrolysis afforded kaempferol in the organic phase, while glucose and rhamnose were identified in the aqueous phase. UV-spectral data in MeOH exhibited 2 characteristic absorption bands at λmax ≈ 346 (I) and ≈ 266 nm (II), confirming a kaempferol nucleus in 1. Bathochromic shift in band (I) with increase in intensity after addition of NaOMe, gave evidence for free 4′-OH. In addition, bathochromic shift in band (II) ( ≈ + 8) upon addition of NaOAc, gave evidence for free 7-OH. The effects of the remaining UV-shift reagents were confirmative for a 5,7,4′-trihydroxy 3-O-glycosylflavonol [25]. Negative ESI-MS revealed a molecular ion peak at m/z 739 [M-H]-, corresponding to Mr 740 of triglycosyl kaempferol with 2 rhamnosyl and one glucosyl moieties. This evidence was further supported by the fragments at m/z 593, 447 and 285, due to the loss of one rhamnosyl, 2 rhamnosyl, then one glucosyl moiety, respectively. 1H NMR spectrum of 1 exhibited an A2X2-spin coupling system of 2 ortho doublets each integrated for 2 protons at 7.90 and 6.84 ppm with J-value of 8.4 Hz, assignable for H-2′/6′ and 3′/5′, respectively indicating a 4′-hydroxy B-ring. An AM-spin coupling system of 2 meta coupled protons at 6.31 and 6.11 ppm of H-8 and H-6, respectively, indicating a 5,7-dihydroxy A-ring. In the aliphatic region, 3 anomeric proton signals were assigned at 5.45 (d, J = 6.9 Hz), 5.01 (brs) and 4.28 (brs) for a β-glucopyranoside and 2 terminal rhamnosyl moieties [23]. The 2 doublet signals of the terminal rhamnosyl methyl groups were observed at 0.92 and 0.76, with J-value of 6.1Hz. 13C NMR spectrum of 1 showed 4 key carbon resonances at 160.3 (C-4′), 133.1 (C-3), 131.4 (C-2′/6′) and 115.8 (C-3′/5′), together with 99.2 (C-6) and 94.6 (C-8), that were confirmative for a kaempferol 3-O-glycoside. In the aliphatic region, there were 18 characteristic signals for one glucosyl and 2 rhamnosyl moieties. The downfield shift of C-4″ of the glucose moiety at 78.3 ( ≈ + 7 ppm) and C-6″ at 69.0 ( ≈ + 7 ppm), were diagnostic evidence for the 2 interglycosidic linkages as (1′′′→4″) and (1″″→6″). All other 13C-resonances were assigned through comparison with previous reported data [24, 26]. Accordingly, compound 1 was identified as kaempferol 3-O-(4″▶  Fig. 1), 6″-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside ( ● which was isolated for the first time from nature. On the basis of its chromatographic properties and UV spectral data, 2 was expected to be a quercetin 3-O-glycoside [25]. Complete acid hydrolysis afforded quercetin in the organic phase, while glucose and rhamnose were identified in the aqueous phase. UV-spectral data in MeOH exhibited 2 characteristic absorption bands at λmax ≈ 357 (I) and ≈ 260 nm (II), to confirm the aglycone as quercetin in compound 2. Bathochromic shift in band (I), with increase in intensity after addition of NaOMe, gave evidence for free 4′-OH. In addition, bathochromic shift in band (II) (≈ + 10 ppm) upon addition of NaOAc, gave evidence for free 7-OH. In ring B, the ortho dihydroxy system was confirmed by the bathochromic shift with NaOAc/H3BO3. The effects of the remaining UV-shift reagents were confirmative for a 5,7,3′,4′-tetrahydroxy 3-O-glycosylflavonol [25]. Negative ESI-MS revealed a molecular ion peak at m/z 755 [M-H]-, corresponding to Mr 756 of triglycosyl quercetin with 2 rhamnosyl and one glucosyl moieties. This evidence was further supported by the fragments at m/z 609, 463 and 301, due to the loss of one rhamnosyl, 2 rhamnosyl and then one glucosyl moiety, respectively. 1H NMR spectrum of 2 exhibited 2 characteristic spin coupling systems in its aromatic region. The first system recorded was an ABM-type of 3 resonances at 7.47 (d, J = 8.4 Hz), a broad meta singlet at 7.40 and an ortho doublet of one proton at 6.74 (d, J = 8.4 Hz), which were assignable to H-6′, H-2′ and H-5′, respectively indicating a

Downloaded by: Chinese University of Hong Kong. Copyrighted material.

262 Original Article

Original Article

5'

6' 8

7 6

9

5

10

OH

1'

O

4

3

O

OH H3C HO

O HO

OH

OH

4''

OH

O

O

O

HO

O

2'

β-D-Glc

O 6''

H3C α-L-Rha HO

HO

3'

2

O

OH β-D-Glc

O α-L-Rha H3C HO

O

HO

O OH

O OH

OH H3C HO

α-L-Rha

O HO

α-L-Rha OH

OH

2

1 OH HO

O

OH

OH HO

O

O OH

O

O OH

HO

OH

OH β-D-Glc

O α-L-Rha H3C HO

O OH OH

OH

O

β-D-Glc

O α-L-Rha H3C HO

Fig. 1  Flavonoids isolated from the aerial parts of A.abyssinicus.

OH

OH

O HO

OH

3

OH OH

4

HO

O

OH

O

5

OH OCH3

3′,4′-dihydroxy B-ring. The second system was an AM-spin coupling system of 2 meta coupled protons at 6.13 and 5.95 of H-8 and H-6, respectively, indicating a 5,7-dihydroxy A-ring. In the aliphatic region, 3 anomeric proton signals were assigned at δ 5.47 (d, J = 7.7 Hz), 5.00 (brs) and 4.30 (brs) for an inner 3-O-βglucopyranoside and 2 terminal α-rhamnosyl moieties [23]. The 2 doublet signals of the terminal rhamnosyl methyl groups were observed at 0.94 and 0.77 with J-value of 6.1 Hz. 13C NMR spectrum of 2 exhibited key carbon resonances at 149.8 (C-4′), 145.5 (C-3′), 132.7 (C-3), 122.0 (C-1′), 121.7 (C-6′), 116.1 (C-2′) and 115.6 (C-5′), which were confirmative for a quercetin 3-O-glycoside. In the aliphatic region there were 18 characteristic signals for one glucosyl and 2 rhamnosyl moieties. The downfield shift of C-4″ of the glucose moiety at 77.7 ( ≈ + 7 ppm) and C-6″ at 68.8 ( ≈ + 6 ppm), were a diagnostic evidence for the 2 interglycosidic linkages as (1′′′→4″) and (1″″→6″), respectively. All other 13C-resonances were assigned through comparison with previous reported data [24, 26]. Accordingly, compound 2 was identified as quercetin 3-O-(4″,6″-di-O-α-L-rhamnopyranosyl)-β-D-gluco­ ▶  Fig. 1), which was isolated for the first time from pyranoside ( ● a natural source. On the basis of its chromatographic properties and UV spectral data, 3 was expected to be a quercetin 3-O-glycoside [25]. Complete acid hydrolysis produced quercetin in the organic phase,

while glucose and rhamnose were identified in the aqueous phase. This compound gave more or less the same UV spectral data of 2, indicating its structure as quercetin 3-O-glycoside [25]. Negative ESI-MS revealed a molecular ion peak at m/z 609 [M-H]-, corresponding to Mr 610 of diglycosyl of one rhamnosyl and one glucosyl moieties. This evidence was further supported by the fragments at m/z 463 and 301, due to the loss of one rhamnosyl and then one glucosyl moiety, respectively. 1H NMR spectrum of 3 exhibited in its aromatic region 2 characteristic spin coupling systems. The first system recorded was a multiplet of 2 protons at 7.48 and an ortho doublet of one proton at 6.79 (J = 8.4 Hz) in the form of an ABM-type, which was assignable to H-2′/6′ and 5′ of a 3′,4′-dihydroxy B-ring. The second coupling system was an AM-spin coupling system of 2 meta coupled protons at 6.34 and 6.14 of H-8 and H-6, respectively, indicating a 5,7-dihydroxy A-ring. In the aliphatic region, 2 anomeric proton signals were assigned at δ 5.29 (d, J = 6.1 Hz) and 4.35 (brs), for a β-glucopyranoside and a terminal rhamnosyl moiety, respectively [23]. In addition, the doublet signal of the terminal rhamnosyl methyl group was observed at 0.94 (d, J = 6.1 Hz).13C NMR spectrum of 3 exhibited key signals for a 3-O-substituted quercetin at 149.0 (C-4′), 145.3 (C-3′), 133.7 (C-3), 122.1 (C-6′), 121.6 (C-1′), 116.7 (C-2′) and 115.7 (C-5′). In the aliphatic region there were 12 characteristic signals for 1 glucosyl and 1 rhamnosyl

El Dib RA et al. Flavonoids from Astragalus abyssinicus  …  Drug Res 2015; 65: 259–265

Downloaded by: Chinese University of Hong Kong. Copyrighted material.

HO

4'

263

264 Original Article

El Dib RA et al. Flavonoids from Astragalus abyssinicus …  Drug Res 2015; 65: 259–265

Fig. 2  SC50 of extracts/compounds isolated from A. abyssinicus in µg/ml. (Color figure available online only).

Conclusion



As far as the available literature is concerned, this is the first report on the chemical composition and bioactivity study of Astragalus abyssinicus. In addition, it is the first report for the isolation of the 2 new flavonoid glycosides, kaempferol 3-O-(4″,6″-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (1) and quercetin 3-O-(4″,6″-di-O-α-L-rhamnopyranosyl)-β-Dglucopyranoside (2) from a natural source. Furthermore, the 3 known compounds quercetin-3-O-(6″-O-α-L-rhamnopyranosyl)β-D-glucopyranoside (3), kaempferol-3-O-(6″-O-α-L-rhamno­ pyranosyl)-β-D-gluco­pyranoside (4) and 5,7,4′-trihydroxy-3′meth­oxyisoflavone (5) were also isolated. All compounds were isolated from the n-BuOH fraction obtained from the 70 % MeOH extract of Astragalus abyssinicus aerial parts (BF), which showed in vitro weak antibacterial activity against Staphylococcus aureus. Since all other tested extracts and compounds did not show any antibacterial activity, the activity of the n-BuOH extract could be attributed to synergistic effect of compounds contained herein. The difference in antioxidant effect of the flavonoid glycosides, being strong for quercetin 3-O-(6″-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (rutin, 3) and lower for quercetin 3-O-(4″,6″-di-O-α-L-rhamno­ pyranosyl)-β-D-glucopyranoside (2) could be attributed to the difference in the number of their sugar units, as they have the same aglycone. However, these results show that Astragalus abyssinicus could be considered as a promising natural antioxidant.

Acknowledgements



This research project was supported by a grant from the “Research Center of the Center for Female Scientific and Medical Colleges”, Deanship of Scientific Research, King Saud University.

Competing Interests



The authors declare that they have no conflict of interests.

Downloaded by: Chinese University of Hong Kong. Copyrighted material.

moieties [24]. The downfield shift of C-6″ at 68.7 (≈ + 8 ppm), was a diagnostic evidence for the interglycosidic linkage as (1′′′→6″). All other 13C-resonances were assigned through comparison with previous reported data of rutin [24, 26]. Accordingly, compound 3 was identified as quercetin 3-O-(6″-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (rutin, ●  ▶  Fig. 1), which was isolated from A. abyssinicus for the first time. Compound 4 was expected to be a flavonyl 3-O-glycoside on the basis of its chromatographic properties and UV spectral data [25]. Complete acid hydrolysis released kaempferol in the organic phase, while glucose and rhamnose were identified in the aqueous phase. UV spectral data, in methanol and all shift reagents, were consistent with that of 1, referring that compound 4 has kaempferol 3-O-glycoside-like structure [25]. 1H NMR spectrum of 4 exhibited an A2X2-spin coupling system of 2 ortho doublets, each integrated for 2 protons at δ 7.93 and 6.83 (J = 8.4), assignable for 2′/6′ and 3′/5′, respectively, indicating a 4′-hydroxyl B-ring. Another AM-spin coupling system of 2 meta coupled protons at 6.34 ppm and 6.13 ppm of H-8 and H-6, respectively, indicated a 5,7-dihydroxy A-ring. In the aliphatic region, 2 anomeric proton signals were assigned at δ 5.25 (d, J = 6.9) and 4.33 (brs) for a β-glucopyranoside and one terminal rhamnosyl moiety, respectively [23]. The doublet signal of the terminal rhamnosyl methyl group was observed at 0.93 (J = 6.1). From UV and 1H NMR spectral data of compound 4, it was identified as kaempferol 3-O-(6″-O-α-L-rhamnopyranosyl)-βD-glucopyranoside (kaempferol 3-O-β-rutinoside), which was isolated for the first time from A. abyssinicus. From the 1H NMR spectrum of 5 it was expected to be an isoflavonoid, due to the assignment of H-2 as a singlet at 8.12 ppm. The appearance of H-6 and H-8 as 2 meta-coupled doublets at 6.22 and 6.34, respectively (J = 1.8), was confirmative for a 5,7-dihydroxy ring A. Also, by comparison with the previously published data, the structure of compound 5 was identified as 5,7,4′-trihydroxy-3′▶  Fig. 1). methoxyisoflavone [23] ( ● Antibacterial activity has ascribed to some Astragalus spp. including A. siculus, A. gummifer and A. melanophrurius. However, this activity has not been investigated for Astragalus abyssinicus before. Therefore, the CHCl3 and the MeOH extracts, as well as extracts resulting from fractionation of the latter, including H2O (WF) and BF, in addition to compounds 2 and 3 were evaluated for their antibacterial activity against the 3 bacteria Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae using paper disc diffusion method. Of all tested extracts or compounds, only BF obtained from the methanol extract showed weak antibacterial activity against Staphylococcus aureus with a zone of inhibition of 7 mm in diameter. Antioxidant activity of the methanol and BF extracts, as well as compounds 2 and 3, was evaluated using ascorbic acid as reference standard and adopting DPPH free radical scavenging activity method. Compound 3 exhibited antioxidant effect (SC50 = 1.29 µg/ml) more than 10 times higher than ascorbic acid (SC50 = 14.56 µg/ml), while compound 2 (SC50 = 9.34 µg/ml) was ▶  Fig. 2). The MeOH more potent than the same reference drug ( ● extract, with SC50 = 38 µg/ml, showed weak antioxidant activity against DPPH. On the other hand, the BF fraction with SC50 = 195 µg/ml did not show any antioxidant activity against DPPH, as compared with ascorbic acid.

References

1 Allen ON, Allen EK. The Leguminosae. A source book of characteristics, uses, and nodulation. First ed. USA: The University of Wisconsin Press, 1981; 72–80 2 Boulos L. Flora of Egypt. First ed. Cairo, Egypt: Alhadara Publishing, 1999; 230–336 3 Verotta L, El-Sebakhy NA. Studies in Natural Products Chemistry: Bioactive Natural Products. Amsterdam: Elsevier, 2001; 179–234 4 Collenette S. Wild flora of Saudi Arabia. First ed. National Commission for Wildlife Conservation and Development (NCWCD), Riyadh, Saudi Arabia: 1999; 479 5 Migahid AM. Flora of Saudi Arabia. Fourth ed. Vol II: Riyadh: University Libraries, King Saud University, 1996; 49 6 Gariboldi P, Pelizzoni F, Tato M et al. Cycloartane triterpene gylcosides from Astragalus trigonus. Phytochemistry 1995; 40: 1755–1760 7 Rios LJ, Waterman GP. A review of the pharmacology and toxicology of Astragalus. Phytother Res 1997; 11: 411–418 8 Bedir E, Pugh N, Calis I et al. Immunostimulatory effects of cycloartanetype triterpene glycosides from Astragalus species. Biol Pharm Bull 2000; 23: 834–837 9 Bedir E, Calis I, Khan IA. Macrophyllosaponin E, A Novel compound from the roots of Astragalus oleifolius. Chem Pharm Bull 2000; 48: 1081–1083 10 Li J, Yu L, Li N et al. Astragalus mongholicus and Angelica sinensis compound alleviates nephrotic hyperlipidemia in rats. Chin Med J 2000; 113: 310–314 11 Lin LZ, He XG, Lindenmaier M et al. Liquid chromatography-electrospray ionization mass spectrometry study of the flavonoids of the roots of Astragalus mongholicus and A. membranaceus. J Chromatogr A 2000; 876: 87–95 12 Wu T, Bligh SWA, Gu L et al. Simultaneous determination of six isoflavonoids in commercial radix Astragali by HPLC-UV. Fitoterapia 2005; 76: 157–165 13 Zhao M, Duan JA, Huang WZ et al. Isoflavans and isoflavone from Astragalus hoantchy. J China Pharm Univ 2002; 33: 274–276

14 Yeom SH, Kim MK, Kim HJ et al. Phenolic compounds from seeds of Astragalus sinicus and its antioxidative activities. Kor J Pharmacogn 2003; 34: 344–351 15 El-sebakhy NA, Assad AM, Abdallah RM et al. Constituents of Egyptian Astragalus tribuloides Del. Nat Prod Sci 2000; 6: 11–15 16 Tang W, Eisenbrand G. Chinese drugs of plant origin, chemistry, pharmacology, and use in traditional and modern medicine. Berlin, Heidelberg: Springer-Verlag, 1992; 377–393 17 Isaev MI, Gorovits MB, Abubakirov NK. Triterpene Glycosides of Astragalus and their Genus, LXV. Cycloartane and Lanostane Triterpenoids of Astragalus orbiculatus. Khim Prir Soedin 1989; 2: 156–175 18 Bisignano G, Iauk L, Kirjavainen S et al. Anti-inflammatory, analgesic, antipyretic and antibacterial activity of Astragalus siculus Biv Int J Pharmacog 1994; 32: 400–405 19 Ross SA, Megalla SE, Bishay DW et al. Studies for determining antibiotic substances in some Egyptian plants. Part I. Screening for antimicrobial activity. Fitoterapia 1980; 51: 303–308 20 Calis I, Yuruker A, Tasdemir D et al. Cycloartan triterpene glycosides from the root of Astragalus melanophrurius. Planta Med 1997; 63: 183–186 21 Harborne JB, Mabry TJ, Mabry H. The Flavonoids. London, New-York: Champman and Hall, 1975 22 Chu YH, Chang CL, Hsu HF. Flavonoid content of several vegetables and their antioxidant activity. J Sci Food Agric 2000; 80: 561–566 23 Harborne JB. The Flavonoids – Advances in Research since 1986. London, New-York: Chapman and Hall, 1994; 116–238 24 Agrawal PK. Carbon-13 NMR of flavonoids. Oxford, New-York: Elsevier, 1989; 96–116 25 Mabry TJ, Markham KR, Thomas MB. The Systematic Identification of Flavonoids. New York: Springer Verlag, 1970 26 Harborne JB, Mabry TJ. The Flavonoids – Advances in Research. London, New-York: Champman and Hall, 1982

El Dib RA et al. Flavonoids from Astragalus abyssinicus  …  Drug Res 2015; 65: 259–265

265

Downloaded by: Chinese University of Hong Kong. Copyrighted material.

Original Article

Two New Flavonoids and Biological Activity of Astragalus abyssinicus (Hochst.) Steud. ex A. Rich. Aerial Parts.

2 new flavonoid glycosides, kaempferol 3-O-(4",6"-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (1) and quercetin 3-O-(4",6"-di-O-α-L-rhamnopyranosyl)...
405KB Sizes 4 Downloads 6 Views