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New flavonol glycosides from Aconitum burnatii Gáyer and Aconitum variegatum L.
Fitoterapia 81 (2010) 940–947
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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
New flavonol glycosides from Aconitum burnatii Gáyer and Aconitum variegatum L. Sara Vitalini a, Alessandra Braca b,⁎, Daniele Passarella c, Gelsomina Fico d a b c d
Dipartimento di Produzione Vegetale, Università degli Studi di Milano, via Celoria 2, 20133 Milano, Italy Dipartimento di Scienze Farmaceutiche, Università di Pisa, Via Bonanno 33, 56126 Pisa, Italy Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, via Venezian 21, 20133 Milano, Italy Dipartimento di Biologia, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy
a r t i c l e
i n f o
Article history: Received 18 January 2010 Received in revised form 4 June 2010 Accepted 13 June 2010 Available online 20 June 2010
Keywords: Aconitum burnatii Aconitum variegatum Ranunculaceae Flavonol glycosides Antioxidant activity
a b s t r a c t Six flavonol glycosides, compounds 1-3 from A. burnatii Gáyer and 4-6 from A. variegatum L., were obtained from their methanol extracts of aerial parts. The identified structures were quercetin 3-O-β- D - glucopyranoside-7-O-(6-E-p-coumaroyl)-β- D -glucopyranosyl(1 → 3)-α- L -rhamnopyranoside (1), quercetin 3-O-β- D- glucopyranoside-7-O-β- D glucopyranosyl-(1 → 3)-α-L-rhamnopyranoside (2), quercetin 3-O-β-D-glucopyranoside-7-O(6-E-caffeoyl)-β- D- glucopyranosyl-(1 → 3)-α- L- rhamnopyranoside (3), kaempferol 3-O-β- D -galactopyranoside-7-O-α- L -arabinopyranoside (4), quercetin 3-O-β- D glucopyranoside (5), and kaempferol 3-O-β-D-glucopyranoside (6). Compounds 1, 2 and 4 were isolated for the first time. The antioxidant potential of the methanol extracts and pure compounds was tested with different assays. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Aconitum (Ranunculaceae) is a genus of herbaceous perennial plants, growing in damp and nitrogenous mountain meadows, distributed on the temperate regions of the northern hemisphere. Aconitum burnatii Gáyer and Aconitum variegatum L. are two European species extending to North and Central Italy, respectively. The first is characterized by simple inflorescence and helmet larger than high; the second shows branched inflorescence and helmet higher than large. Both species are characterized by tuberous roots, alternate and palmately leaves, blue-violet flowers, brownish seeds and follicles as fruits [1–4]. The high variability of Aconitum genus morphologic traits has always attracted the attention of botanists. Many taxonomic studies [1–3,5,6] have been performed to clarify the systematic of the genus that appears particularly complex; in the last years, researches on its alkaloids and flavonoids as ⁎ Corresponding author. Tel.: + 39 050 2219688; fax: + 39 050 2219660. E-mail address: [email protected] (A. Braca). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.06.012
chemotaxonomic markers have given further information to characterize the Aconitum species [7–9]. Roots, leaves and flowers, riches in these compounds, were employed in oriental medicine [10–12]. Chinese people, for example, apply (external use) Aconitum aerial parts to treat abrasions and wounds. For internal use, despite the high toxicity of this genus, inducing physiological responses as increased salivation, respiratory paralysis, muscular weakness and convulsions, it was historically employed as antirheumatic, antineuralgic, to warm up the body and ease pain. A particular use of the roots is in a soup with different kinds of meat and vegetables [13]. In all these cases, the detoxification is bound to a boiling (hydrolysis procedure) by the decomposition of the diester diterpene alkaloids to the less toxic monoester diterpene alkaloids. Alkaloids and flavonoids of this genus were studied for their pharmacological activities [9,13–16]. In particular, the alkaloid composition of A. variegatum and A. burnatii, characterized by norditerpene and diterpene compounds, has been already investigated [17–22], while nothing is reported about their flavonoid content. In this work, for the first time, their flavonoid composition and in vitro antioxidant activity have been studied.
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2. Experimental 2.1. General methods Optical rotations were measured on a Perkin-Elmer 241 polarimeter equipped with a sodium lamp (589 nm) and a 1 dm microcell. UV spectra were recorded on a Perkin-Elmer Lambda 11 spectrometer. NMR analysis was performed on a Bruker 400 MHz spectrometer at 300 K; chemical shifts are expressed in δ (parts per million) referenced to the solvent peaks δH 3.34 and δC 49.0 for CD3OD. The standard pulse sequence was used for 1D TOCSY, DQF-COSY, HSQC, and HMBC experiments. HRESIMS spectra were recorded on FTICR APEXII (Bruker Daltonics). ESIMS were obtained from an LCQ Advantage ThermoFinnigan spectrometer, equipped with a Xcalibur software. HPLC separation was performed on a Merck Hitachi L-6200 Intelligent Pumping system, with a Merck Hitachi L-4200 UV–VIS detector, a Waters μ-Bondapak C18 column and LC Organizer injector. TLC was conducted on silica 60 F254 gel-coated aluminium sheets (Merck). GC analyses were performed using a Dani GC 1000 instrument on a L-CP-Chirasil-Val column (0.32 mm × 25 m). 2.2. Plant material and morphological analysis The Aconitum species were collected during the summer of 2006 in the North of Italy, on the Cozie and Retiche Alps: A. burnatii at Colle di Sampeyre (2300 m), Cuneo and A. variegatum at Bormio (1250 m), Sondrio respectively. In order to correctly identify the species under study, samples (10), both flowers (30) and leaves (30) collected from different plants were analysed. The distinguishing characters (glandular hairs on the whole plant for A. burnatii; follicles hairy at the suture and nectary straight, not curved forward, for A. variegatum) were chosen on the basis of literature data [1,3,4]. Both in vivo and exsiccata samples of leaves and flowers were observed with a stereomicroscope (LEICA MZ6). Voucher specimens (A. burnatii no. Ab 201–210; A. variegatum no. Av 211–220) were deposited at the Herbarium of Department of Biology, University of Milan. 2.3. Extraction and isolation Dried and powdered aerial parts of A. burnatii (55 g) and A. variegatum (64 g) were defatted with n-hexane and successively extracted with CHCl3, CHCl3:MeOH (9:1), and MeOH. A. burnatii. The methanol extract (4.0 g) was chromatographed on Sephadex LH-20, using MeOH as eluent, to obtain 320 fractions of 3 ml, combined together into 24 groups, according to TLC separations [Silica 60 F254-gel coated aluminium sheets; eluent: n-BuOH–CH 3 COOH–H 2 O (60:15:25)]. Group 8 was submitted to RP-HPLC on C18 μBondapak column (300 × 7.8 mm, flow rate 2.5 ml min−1) with MeOH–H2O (40:60) to yield compounds 2 (t R = 15 min, 5 mg) and 1 (t R = 26 min, 5.7 mg). Crystallization of group 10 from MeOH yielded compound 3 (64.9 mg). A. variegatum. The methanol extract (3.3 g) was chromatographed on Sephadex LH-20, using MeOH as eluent, to obtain 166 fractions of 3 ml, combined together into 25 groups, according to TLC separations [Silica 60 F254-gel coated aluminium sheets; eluent: n-BuOH–CH 3 COOH–H 2 O
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(60:15:25)]. Groups 5 to 9 were submitted to RP-HPLC on C18 μ-Bondapak column (300 ×7.8 mm, flow rate 2.5 ml min−1) with MeOH–H2O (40:60) to yield 4 (t R = 19 min, 4 mg). Crystallization of groups 23 and 24 from MeOH yielded compounds 5 (8 mg) and 6 (14 mg), respectively. 2.3.1. Quercetin 3-O-β- D- glucopyranoside-7-O-(6-E-pcoumaroyl)-β-D-glucopyranosyl-(1→ 3)-α-L-rhamnopyranoside (1) Yellow amorphous powder, [α]D: 58° (c 0.1, MeOH); UV λmax (MeOH) nm (log ε): 255 (3.15), 320 (3.97); 1H and 13C NMR (400 and 100 MHz, CD3OD): see Table 1; HRESIMS: m/z 917.23510 [M–H]− (calcd for C42H46O23, 918.24299); ESIMS: m/z 917 [M–H]−, 755 [M–H–162]−, 609 [M–H–162–146]−, 301 [M-H-162-146-162-146]−. 2 . 3 . 2 . Quercetin 3-O-β- D - glucopyranoside-7-O-β- D glucopyranosyl-(1→ 3)-α-L-rhamnopyranoside (2) Yellow amorphous powder, [α]D: 45° (c 0.1, MeOH); UV λmax (MeOH) nm (log ε): 256 (3.89), 325 sh (3.51), 360 (3.76); 1H and 13C NMR (400 and 100 MHz, CD3OD): see Table 1; HRESIMS: m/z 771.19868 [M–H]− (calcd for C33H40O21, 772.20621); ESIMS: m/z 771 [M–H]−, 609 [M– H–162]−, 463 [M–H–162–146]−, 301 [M–H–162–146–162]−. 2.3.3. Kaempferol 3-O-β- D -galactopyranoside-7-O-α- L arabinopyranoside (4) Yellow amorphous powder, [α]D: -32° (c 0.1, MeOH); UV λmax (MeOH) nm (log ε): 265 (3.78), 348 (3.71); 1H and 13C NMR (400 and 100 MHz, CD3OD): see Table 1; HRESIMS: m/z 603.12970 [M + Na]+ (calcd for C26H28O15, 580.14282); ESIMS: m/z 579 [M–H]−, 447 [M–H–132]−, 285 [M–H–132– 162]−. 2.3.4. Acid hydrolysis of compounds 1, 2, and 4 A solution of each compound (2.0 mg) in 1 N HCl (1 ml) was stirred at 80 °C in a stoppered reaction vial for 4 h. After cooling, the solution was evaporated under a stream of N2. Each residue was dissolved in 1-(trimethylsilyl)imidazole and pyridine (0.2 ml), and the solution was stirred at 60 °C for 5 min. After drying, the residue was partitioned between H2O and CHCl3. The CHCl3 layer was analyzed by GC using a L-CP-Chirasil-Val column (0.32 mm × 25 m). Temperatures of both the injector and detector were 200 °C. A temperature gradient system was used for the oven, starting at 100 °C for 1 min and increasing up to 180 °C at a rate of 5 °C/min. Peaks of the hydrolysate were detected by comparison with retention times of authentic samples of D-glucose, D-galactose, L-arabinose, and L-rhamnose (Sigma Aldrich), after treatment with 1-(trimethylsilyl)imidazole in pyridine. 2.4. Antioxidant activity 2.4.1. Determination of polyphenolic content Total polyphenols of the extracts were quantified colorimetrically by the Folin-Ciocalteau assay, using gallic acid as standard [23]. Briefly, a suitable aliquot of the sample was combined with 50 μl of Folin-Ciocalteau reagent;3 min, 100 μl of a saturated sodium carbonate solution was added and the final volume (2.5 ml) was reached with distilled water. After 1 h of incubation in the dark at room temperature, the
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absorbance was read at 725 nm and molarity was referred to as gallic acid equivalents. 2.4.2. DPPH scavenging test An appropriate aliquot of the extracts or pure compounds (at concentrations ranging from 1 to 100 μM) was added to a 15 μM ethanol solution of DPPH (2,2-diphenyl-1-picrylhydrazil radical). The samples were incubated in the dark for 15 min and
their absorbance was measured at 517 nm [24]. The assay was performed in triplicate and the IC50 was calculated with Prism® 4 (GraphPad Software Inc.) software. 2.4.3. Total antioxidant capacity The total antioxidant capacity of the samples was determined on the basis of the copper reducing (Cu++ to Cu+) ability by a validated method (BIOXYTECH® AOP-490™, Oxis
Table 1 1 H and 13C NMR data of compounds 1, 2, and 4 (CD3OD, 400 and 100 MHz, J in Hz, δ in ppm) a. 1 δΗ 2 3 4 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' 3-O-Glc 1 2 3 4 5 6a 6b 7-O-Rha 1 2 3 4 5 6 Glc 1 2 3 4 5 6a 6b COO α β 1 2/6 3/5 4 3-O-Gal 1 2 3 4 5 6a 6b 7-O-Ara 1 2 3 4 5a 5b a
6.32 d (2.0) 6.33 d (2.0)
7.72 d (2.0)
6.90 d (8.5) 7.63 dd (8.5, 2.0) 5.32 d (7.5) 3.49 dd (9.0, 7.5) 3.50 t (9.0) 3.33 t (9.0) 3.40 m 3.80 dd (12.0, 3.0) 3.60 dd (12.0, 5.0) 5.41 d (1.8) 4.32 dd (3.0, 1.8) 3.84 dd (9.0, 3.0) 3.65 t (9.0) 3.60 m 1.22 d (6.5) 4.54 d (7.5) 3.42 dd (9.0, 7.5) 3.31 t (9.0) 3.43 t (9.0) 3.68 m 4.54 dd (12.0, 3.0) 4.52 dd (12.0, 5.0) 6.25 d (16.0) 7.54 d (16.0) 7.13 d (8.0) 6.44 d (8.0)
2 δΧ 158.0 135.5 179.4 162.5 100.0 163.5 95.5 156.8 105.0 122.8 117.1 145.8 149.0 115.9 123.3 104.0 75.5 77.9 71.3 78.1 62.5 99.0 71.1 83.4 72.0 70.6 18.0 106.1 75.3 78.0 71.0 75.5 64.8
δΗ
6.49 d (1.8) 6.78 d (1.8)
7.72 d (2.0)
6.85 d (8.5) 7.63 dd (8.5, 2.0) 5.31 d (7.5) 3.45 dd (9.0, 7.5) 3.50 t (9.0) 3.33 t (9.0) 3.38 m 3.80 dd (12.0, 2.5) 3.65 dd (12.0, 4.5) 5.60 d (1.8) 4.32 dd (3.0, 1.8) 3.94 dd (9.0, 3.0) 3.65 t (9.0) 3.66 m 1.28 d (6.5) 4.63 d (7.5) 3.49 dd (9.0, 7.5) 3.38 t (9.0) 3.42 t (9.0) 3.40 m 3.84 dd (12.0, 3.0) 3.60 dd (12.0, 5.0)
4 δΧ 158.2 135.2 179.5 162.5 100.5 163.6 95.5 156.9 105.2 122.5 116.9 146.0 148.7 116.1 123.6 104.3 75.4 77.9 71.2 77.8 62.5
δΗ
6.45 d (1.8) 6.73 d (1.8)
8.12 d (8.0) 6.93 d (8.0) 6.93 d (8.0) 8.12 d (8.0)
δΧ 157.8 135.2 178.3 163.1 98.9 165.4 96.5 159.3 104.5 123.0 132.2 116.2 161.5 116.2 132.2
99.6 71.0 82.5 72.2 70.8 18.0 105.8 75.4 77.7 71.0 77.6 62.5
168.9 114.4 146.8 126.8 131.0 116.2 161.0
Assignments were confirmed by DQF-COSY, 1D-TOCSY, HSQC, and HMBC experiments.
5.58 d (7.8) 3.71 dd (9.0, 7.8) 3.49 dd (9.0, 4.0) 3.65 dd (4.0, 2.5) 3.60 m 3.80 dd (12.0, 2.5) 3.50 dd (12.0, 4.5) 5.02 d (7.0) 3.91 dd (9.0, 7.0) 3.49 dd (9.0, 2.5) 3.70 m 3.90 dd (12.0, 2.0) 3.63 dd (12.0, 3.0)
101.7 72.9 77.5 73.5 77.3 65.3 106.4 72.1 78.0 73.1 65.4
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Research™, Portland, OR) [25]. The samples were tested at concentrations of 10 and 1 μM. The results were shown as mEq uric acid. 3. Results and discussion 3.1. Morphological analysis In order to evaluate the morphologic traits more suitable for the species determination, four taxonomic keys [1–4] have been used. In particular, ten characters were used as diacritic for
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A. burnatii: the roots of the entities appear tuberous; the leaves are divided with linear segments not more than 3 mm wide; the plant stalk is straight; the inflorescence is simple; the flowers are blue-violet; the ratio height/width of helmet is up to 0.7; only glandular hairs along the inflorescence are densely present; the nectaries appear bent forward; the fruits are glabrous follicles; the seeds present three smooth or rugolose sides with three winged angles, one larger than other two. The same number of characters allowed the A. variegatum identification: the roots are tuberous; the leaves are divided to the base and their segments, more than 3 mm wide, are often
Fig. 1. A. burnatii Gáyer.
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Fig. 2. A. variegatum L.
divided to beyond the middle and deeply incise-dentate; the plant stalk has a zigzag line; the inflorescence is branched; the flowers are blue or variegated with helmet about twice as high as wide; only eglandular hairs along the inflorescence are present; the nectaries appear straight; the follicles are hairy at the sutures; the seeds are winged on one angle with prominent transverse lamellae on the sides. Figs. 1 and 2 show the discriminant characters of two species under study, useful for their identification. On the basis of morphological data, the population from Colle di Sampeyre (Cuneo) and Bormio (Sondrio), Italy, can be ascribed to the species Aconitum burnatii and A. variegatum, respectively.
3.2. Phytochemical study The methanol extracts of A. burnatii and A. variegatum aerial parts were chromatographed on Sephadex LH-20 and RP-HPLC (C-18) to yield pure compounds 1–6 (Figs. 3 and 4).
Compound 1, a yellow amorphous solid, showed molecular formula C42H46O23 by means of HRESIMS ([M-H]− peak at m/z 917.23510). Its ESIMS spectrum showed peaks at m/z 755 [M–H–162]−, 609 [M–H–162–146]−, and 301 [M–H– 162–146–162–146]− due to the loss of two hexoses, one deoxyhexoses, and one p-coumaroyl residue. The UV spectrum of 1 showed two absorption maxima at 320 and 255 nm, indicating the presence of substituted aromatic rings and α,β unsaturated ketone in the molecule. The 1H and 13C NMR spectra of compound 1 (Table 1) showed a typical pattern of a flavonol with quercetin aglycon and signals ascribable to sugar moieties and acyl residue [26]. The three anomeric protons arising from the sugar moieties appeared at δ 5.41 (1H, d, J = 1.8 Hz), 5.32 (1H, d, J = 7.5 Hz), and 4.54 (1H, d, J = 7.5 Hz), which correlated respectively with signals at 99.0, 104.0, and 106.1 in the HSQC spectrum. The 1H NMR showed also the presence of a p-coumaroyl residue (Table 1). Assignments of all chemical shifts of protons and carbons of the aglycon, sugar, and acyl portions were ascertained from a
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Fig. 3. Structures of flavonol glycosides 1–3 isolated from A. burnatii.
combination of 1D-TOCSY, DQF-COSY, and HSQC analysis. The configurations of the sugar units were assigned after hydrolysis of 1 with 1 N HCl. The hydrolysate was trimethyl-
silylated and GC retention times were compared with those of authentic sugar samples prepared in the same manner. The lower field shifts of H-6a and H-6b (δ 4.52, 4.54) and C-6
Fig. 4. Structures of flavonol glycosides 4–6 isolated from A. variegatum.
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Table 2 Antioxidant activity of A. burnatii and A. variegatum crude extracts and flavonols. Samples
Polyphenols (mg/g extract)
Ascorbic acid TroloxTM Quercetin
DPPH (IC50) a
Antioxidant capacity (mEq uric acid) b
μM
1 μM
10 μM
9.25 ± 0.89 12.9 ± 0.95 4.37 ± 0.21
0.36 ± 0.02 0.85 ± 0.07 0.75 ± 0.06
0.33 ± 0.02 1.77 ± 0.18 2.17 ± 1.35
A. burnatii extract 1 2 3
173.45 ± 1.58
13.2 ± 0.76 11.8 ± 0.54 12.1 ± 0.55 2.02 ± 0.24
0.37 ± 0.12 0.10 ± 0.09 0.09 ± 0.03 0.15 ± 0.07
1.49 ± 0.65 0.54 ± 0.07 0.46 ± 0.06 0.81 ± 0.11
A. variegatum extract 4 5 6
113.47 ± 1.32
26.7 ± 0.67 356 ± 1.11 295 ± 1.02 277 ± 1. 19
0.12 ± 0.09 0.04 ± 0.02 0.04 ± 0.01 0.05 ± 0.01
1.08 ± 0. 23 0.06 ± 0.01 0.33 ± 0.04 0.46 ± 0.06
a
IC50 = sample concentration required for 50% DPPH free radical scavenging. mEq uric acid = unit of measurement of the antioxidant capacity for copper reduction. Experiments were performed in triplicate and results are mean ± SD. Ascorbic acid, Trolox®, and quercetin are the standard references. b
(64.8) signals of one glucosyl unit, together with the lower field shifts of C-3 signal of rhamnose unit (83.4 ppm), suggested the substitution pattern of glycosyl and acyl moieties. The substitution sites on the molecule were confirmed by an HMBC experiment, showing correlation peaks between δ 5.32 (H-1glcI) and 135.5 (C-3), δ 4.54 (H1glcII) and 83.4 (C-3rha), δ 5.41 (H-1rha) and 163.5 (C-7), δ 4.52 and 4.54 (H-6glcII) and 168.9 (COO). From these results, the structure of 1 was determined to be quercetin 3-O-β-Dglucopyranoside-7-O-(6-E-p-coumaroyl)-β-D-glucopyranosyl-(1 → 3)-α-L-rhamnopyranoside. Compound 2 was obtained as a yellow amorphous powder, with the molecular formula C33H40O21, as deduced from the [MH]− peak at m/z 771.19868 by HRESIMS and confirmed by 13C and 13C DEPT data. The ESIMS of compound 2 showed a prominent fragment at m/z 771 [M-H]− and a fragmentation pattern similar to that of 1. The spectroscopic data of the aglycone moiety of 2 were identical to those of 1. The proton coupling network within each sugar residue was established, using a combination of 1D TOCSY, DQF-COSY, and HSQC experiments. Again, direct evidence for the sugar sequence and the linkage sites was derived from the HSQC and HMBC data. Comparison of NMR data of the sugar moiety (Table 1) of 2 with that of 1 indicated that 2 differed from 1 only by the absence of the p-coumaroyl moiety. The configuration of the sugar units was determined as reported for compound 1. Thus, compound 2 was defined as quercetin 3-O-β-D-glucopyranoside-7-O-β-D-glucopyranosyl-(1 → 3)-α-L-rhamnopyranoside (2). The molecular formula C26H28O15 was assigned to compound 4 as determined by HRESIMS ([M + Na]+ at m/z 603.12970). The UV spectrum was similar to those of 1, suggesting a flavonol glycoside structure. The 1H and 13C NMR of 4 (Table 1) were consistent with a kaempferol glycoside with signals ascribable to aglycon portion and sugar moieties. The two anomeric protons arising from the sugar residues appeared at δ 5.58 (1H, d, J = 7.8 Hz) and 5.02 (1H, d, J = 7.0 Hz) which correlated respectively with signals at 101.7 and 106.4 ppm in the HSQC spectrum. Assignments of all chemical shifts of protons and carbons of the aglycon and sugar
portions were ascertained from a combination of 1D-TOCSY, DQF-COSY, and HSQC analysis. The configurations of the sugar units were assigned after hydrolysis of 4 with 1 N HCl. The hydrolysate was trimethylsilylated and GC retention times were compared with those of authentic sugar samples prepared in the same manner. The substitution sites on the molecule were confirmed by an HMBC experiment showing correlation peaks between δ 5.58 (H-1gal) and 135.5 (C-3), δ 5.02 (H-1ara) and 165.4 (C-7). From these results, the structure of 1 was determined to be kaempferol 3-O-β-D-galactopyranoside-7-Oα-L-arabinopyranoside. Three known flavonols, quercetin 3-O-β-D-glucopyranoside-7-O-(6-E-caffeoyl)-β- D- glucopyranosyl-(1 → 3)-α- Lrhamnopyranoside (3), quercetin 3-O-β-D-glucopyranoside (5), and kaempferol 3-O-β-D-glucopyranoside (6) were also isolated and identified by spectral analysis and comparison of data with those reported in the literature [26,27]. Compound 3 was formerly identified in A. napellus ssp. neomontanum (Wulfer) Gáyer [27], while 5 and 6 were common flavonol glycosides characterized also from A. jaluense complex [7]. 3.3. Antioxidant activity The methanolic extracts obtained from A. burnatii and A. variegatum respectively, were tested for their polyphenolic content by the Folin-Ciocalteau method. The highest concentration of polyphenols was found in A. burnatii (Table 2). The two crude extracts were also analyzed to assess their antioxidant potential by employing different in vitro assays, i.e., DPPH radical scavenging test and total antioxidant capacity based upon the reduction of Cu++ to Cu+. In both assays, ascorbic acid, Trolox®, and quercetin were used as standards. As shown in Table 2, A. burnatii extract displayed a higher activity in comparison with A. variegatum residue. It exhibited a removal action of the stable radical DPPH twice stronger than that of A. variegatum extract. This action was similar to that of ascorbic acid and Trolox®, but three times lower than that of quercetin. Concerning overall antioxidant capacity, activity was noted, for both samples, at concentration of 10 μM (Table 2). From these extracts, compounds 1–6
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were isolated and then tested for their antioxidant power (Table 2). The scavenging abilities of 1 and 2 were comparable with those of vitamin C and Trolox®, compound 3 showed a value similar to quercetin, in line with literature data. The catechol structure conferred high stability to the radical form and participated in the electron delocalisation. The caffeoyl derivative 3 had a higher radical scavenging ability than the p-coumaroyl ester 1 [16]. The presence of the caffeoyl moiety enhances the activity of 3 in comparison with 5 [9]. Compounds 4, 5, and 6 were poorly active. All six molecules exhibited limited antioxidant capacity at 1 μM which increased at 10 μM, particularly for 3. Acknowledgements The authors thank Dr. Cristina Mariani for photo presentation and Dr. Marcello Iriti for his cooperation. References [1] Seitz W. Die Taxonomie der Aconitum napellus-Gruppe in Europa. Feddes Repertorium 1969;80:1–76. [2] Hegi G. Illustrierte Flora von Mitteleuropa. München: Carl Hansen; 1974. p. 153–77. [3] Pignatti S. Flora d'Italia, vol. 1. Bologna: Edagricole; 1982. p. 285–8. [4] Akeroyd JR, Charter AO, Aconitum L. In: Tutin TG, Burges NA, Charter AO, Edmondson JR, Heywood VH, Moore DM, Valentie DH, Walters SM, Webb DA, editors. Flora Europaea. 2nd Eds. Cambridge: Cambridge University Press; 1993. p. 254–6. [5] Tutin TG, Burges NA, Charter AO, Edmondson JR, Heywood VH, Moore DM, Valentie DH, Walters SM, Webb DA. Flora Europaea. Cambridge: Cambridge University Press; 1993. [6] Mücher W. Die gattung Aconitum in Kärnten. Carinthia II 1993;183/103: 519–27. [7] Lim CE, Park JK, Park CW. Flavonoid variation of the Aconitum jaluense complex (Ranunculaceae) in Korea. Plant Syst Evol 1999;218:125–31. [8] Fico G, Spada A, Braca A, Agradi E, Morelli I, Tomè F. RAPD analysis and flavonoid composition of Aconitum as an aid for taxonomic discrimination. Bioch Syst Ecol 2003;31:293–301. [9] Mariani C, Braca A, Vitalini S, De Tommasi N, Visioli F, Fico G. Flavonoid characterization and in vitro antioxidant activity of Aconitum anthora L. (Ranunculaceae). Phytochemistry 2008;69:1220–6.
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[10] Schauenberg P, Paris F. Plants containing alkaloids. Guide to medicinal plants. London: Lutterworth Press; 1977. [11] Bisset NG. Arrow poisons in China. Part II. Aconitum - botany, chemistry and pharmacology. J Ethnopharmacol 1981;4:247-36. [12] Ikram M, Gul K, Gilani SN. Antipyretic studies on some indigenous Pakistani medicinal plants: II. J Ethnopharmacol 1987;19:185–92. [13] Singhuber J, Zhu M, Prinz S, Kopp B. Aconitum in traditional Chinese medicine — a valuable drug or an unpredictable risk? J Ethnopharmacol 2009;126:18–30. [14] Feldkamp A, Köster B, Weber HP. Fatal poisoning by monkshood (Aconitum napellus). Monatsschr Kinderh 1991;139:366–7. [15] Chan T. Aconitine poisoning: a global perspective. Vet Hum Toxicol 1994;36:326–8. [16] Braca A, Fico G, Morelli I, De Simone F, Tomè F, De Tommasi N. Antioxidant and free radical scavenging activity of flavonol glycosides from different Aconitum species. J Ethnopharmacol 2003;86:63–7. [17] Khaimova MA, Palamareva MD, Grozdanova LG, Mollov NM. Panov PP Alkaloids of Aconitum variegatum, a common plant in Bulgaria. CR Acad Bulg Sci 1967;20:193–6. [18] Mody NV, Pelletier SW, Mollov NM. The structure of cammaconine from Aconitum variegatum. Heterocycles 1980;14:1751–2. [19] Staehelin E, Katz A. Thin layer chromatographic screening of European Aconitum variegatum spp. as well as the isolation and partial formulation of paniculatin. Pharm Acta Helv 1980;55:221–7. [20] Katz A, Rudin HP, Staehelin E. Aconitum. Isolation of talatisamine and 14-O-acetyltalatisamine from the tubers of Aconitum paniculatum Lam., Aconitum toxicum Rchb., and of the subspecies nasutum Goetz and pyrenaicum Vivant and Delay of Aconitum variegatum L. The alkaloids of the seeds of Aconitum paniculatum Lam. Pharm Acta Helv 1987;62:216–20. [21] Díaz JG, Ruiza JG, Herz W. Norditerpene and diterpene alkaloids from Aconitum variegatum. Phytochemistry 2005;66:837–46. [22] Katz A, Staehelin E. TLC study on the European Aconitum napellus group. Pharm Acta Helv 1979;54:253–65. [23] Visioli F, Vinceri FF, Galli C. Waste waters from olive oil production are rich in natural antioxidants. Experientia 1995;51:32–4. [24] Visioli F, Galli C. The effect of minor constituents of olive oil on cardiovascular disease: new findings. Nutr Rev 1998;56:142–7. [25] Visioli F, Caruso D, Plasmati E, Patelli R, Mulinacci N, Romani A, Galli G, Galli C. Hydroxytyrosol, as a component of olive mill wastewater, is dose-dependently absorbed and increases the antioxidant capacity of rat plasma. Free Radic Res 2001;34:301–5. [26] Agrawal PK. Carbon-13 NMR of flavonoids. Amsterdam: Elsevier Science Publishers; 1989. [27] Fico G, Braca A, De Tommasi N, Tomè F, Morelli I. Flavonoids from Aconitum napellus subsp. neomontanum. Phytochemistry 2001;57:543–6.
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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
New flavonol glycosides from Aconitum burnatii Gáyer and Aconitum variegatum L. Sara Vitalini a, Alessandra Braca b,⁎, Daniele Passarella c, Gelsomina Fico d a b c d
Dipartimento di Produzione Vegetale, Università degli Studi di Milano, via Celoria 2, 20133 Milano, Italy Dipartimento di Scienze Farmaceutiche, Università di Pisa, Via Bonanno 33, 56126 Pisa, Italy Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, via Venezian 21, 20133 Milano, Italy Dipartimento di Biologia, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy
a r t i c l e
i n f o
Article history: Received 18 January 2010 Received in revised form 4 June 2010 Accepted 13 June 2010 Available online 20 June 2010
Keywords: Aconitum burnatii Aconitum variegatum Ranunculaceae Flavonol glycosides Antioxidant activity
a b s t r a c t Six flavonol glycosides, compounds 1-3 from A. burnatii Gáyer and 4-6 from A. variegatum L., were obtained from their methanol extracts of aerial parts. The identified structures were quercetin 3-O-β- D - glucopyranoside-7-O-(6-E-p-coumaroyl)-β- D -glucopyranosyl(1 → 3)-α- L -rhamnopyranoside (1), quercetin 3-O-β- D- glucopyranoside-7-O-β- D glucopyranosyl-(1 → 3)-α-L-rhamnopyranoside (2), quercetin 3-O-β-D-glucopyranoside-7-O(6-E-caffeoyl)-β- D- glucopyranosyl-(1 → 3)-α- L- rhamnopyranoside (3), kaempferol 3-O-β- D -galactopyranoside-7-O-α- L -arabinopyranoside (4), quercetin 3-O-β- D glucopyranoside (5), and kaempferol 3-O-β-D-glucopyranoside (6). Compounds 1, 2 and 4 were isolated for the first time. The antioxidant potential of the methanol extracts and pure compounds was tested with different assays. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Aconitum (Ranunculaceae) is a genus of herbaceous perennial plants, growing in damp and nitrogenous mountain meadows, distributed on the temperate regions of the northern hemisphere. Aconitum burnatii Gáyer and Aconitum variegatum L. are two European species extending to North and Central Italy, respectively. The first is characterized by simple inflorescence and helmet larger than high; the second shows branched inflorescence and helmet higher than large. Both species are characterized by tuberous roots, alternate and palmately leaves, blue-violet flowers, brownish seeds and follicles as fruits [1–4]. The high variability of Aconitum genus morphologic traits has always attracted the attention of botanists. Many taxonomic studies [1–3,5,6] have been performed to clarify the systematic of the genus that appears particularly complex; in the last years, researches on its alkaloids and flavonoids as ⁎ Corresponding author. Tel.: + 39 050 2219688; fax: + 39 050 2219660. E-mail address: [email protected] (A. Braca). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.06.012
chemotaxonomic markers have given further information to characterize the Aconitum species [7–9]. Roots, leaves and flowers, riches in these compounds, were employed in oriental medicine [10–12]. Chinese people, for example, apply (external use) Aconitum aerial parts to treat abrasions and wounds. For internal use, despite the high toxicity of this genus, inducing physiological responses as increased salivation, respiratory paralysis, muscular weakness and convulsions, it was historically employed as antirheumatic, antineuralgic, to warm up the body and ease pain. A particular use of the roots is in a soup with different kinds of meat and vegetables [13]. In all these cases, the detoxification is bound to a boiling (hydrolysis procedure) by the decomposition of the diester diterpene alkaloids to the less toxic monoester diterpene alkaloids. Alkaloids and flavonoids of this genus were studied for their pharmacological activities [9,13–16]. In particular, the alkaloid composition of A. variegatum and A. burnatii, characterized by norditerpene and diterpene compounds, has been already investigated [17–22], while nothing is reported about their flavonoid content. In this work, for the first time, their flavonoid composition and in vitro antioxidant activity have been studied.
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2. Experimental 2.1. General methods Optical rotations were measured on a Perkin-Elmer 241 polarimeter equipped with a sodium lamp (589 nm) and a 1 dm microcell. UV spectra were recorded on a Perkin-Elmer Lambda 11 spectrometer. NMR analysis was performed on a Bruker 400 MHz spectrometer at 300 K; chemical shifts are expressed in δ (parts per million) referenced to the solvent peaks δH 3.34 and δC 49.0 for CD3OD. The standard pulse sequence was used for 1D TOCSY, DQF-COSY, HSQC, and HMBC experiments. HRESIMS spectra were recorded on FTICR APEXII (Bruker Daltonics). ESIMS were obtained from an LCQ Advantage ThermoFinnigan spectrometer, equipped with a Xcalibur software. HPLC separation was performed on a Merck Hitachi L-6200 Intelligent Pumping system, with a Merck Hitachi L-4200 UV–VIS detector, a Waters μ-Bondapak C18 column and LC Organizer injector. TLC was conducted on silica 60 F254 gel-coated aluminium sheets (Merck). GC analyses were performed using a Dani GC 1000 instrument on a L-CP-Chirasil-Val column (0.32 mm × 25 m). 2.2. Plant material and morphological analysis The Aconitum species were collected during the summer of 2006 in the North of Italy, on the Cozie and Retiche Alps: A. burnatii at Colle di Sampeyre (2300 m), Cuneo and A. variegatum at Bormio (1250 m), Sondrio respectively. In order to correctly identify the species under study, samples (10), both flowers (30) and leaves (30) collected from different plants were analysed. The distinguishing characters (glandular hairs on the whole plant for A. burnatii; follicles hairy at the suture and nectary straight, not curved forward, for A. variegatum) were chosen on the basis of literature data [1,3,4]. Both in vivo and exsiccata samples of leaves and flowers were observed with a stereomicroscope (LEICA MZ6). Voucher specimens (A. burnatii no. Ab 201–210; A. variegatum no. Av 211–220) were deposited at the Herbarium of Department of Biology, University of Milan. 2.3. Extraction and isolation Dried and powdered aerial parts of A. burnatii (55 g) and A. variegatum (64 g) were defatted with n-hexane and successively extracted with CHCl3, CHCl3:MeOH (9:1), and MeOH. A. burnatii. The methanol extract (4.0 g) was chromatographed on Sephadex LH-20, using MeOH as eluent, to obtain 320 fractions of 3 ml, combined together into 24 groups, according to TLC separations [Silica 60 F254-gel coated aluminium sheets; eluent: n-BuOH–CH 3 COOH–H 2 O (60:15:25)]. Group 8 was submitted to RP-HPLC on C18 μBondapak column (300 × 7.8 mm, flow rate 2.5 ml min−1) with MeOH–H2O (40:60) to yield compounds 2 (t R = 15 min, 5 mg) and 1 (t R = 26 min, 5.7 mg). Crystallization of group 10 from MeOH yielded compound 3 (64.9 mg). A. variegatum. The methanol extract (3.3 g) was chromatographed on Sephadex LH-20, using MeOH as eluent, to obtain 166 fractions of 3 ml, combined together into 25 groups, according to TLC separations [Silica 60 F254-gel coated aluminium sheets; eluent: n-BuOH–CH 3 COOH–H 2 O
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(60:15:25)]. Groups 5 to 9 were submitted to RP-HPLC on C18 μ-Bondapak column (300 ×7.8 mm, flow rate 2.5 ml min−1) with MeOH–H2O (40:60) to yield 4 (t R = 19 min, 4 mg). Crystallization of groups 23 and 24 from MeOH yielded compounds 5 (8 mg) and 6 (14 mg), respectively. 2.3.1. Quercetin 3-O-β- D- glucopyranoside-7-O-(6-E-pcoumaroyl)-β-D-glucopyranosyl-(1→ 3)-α-L-rhamnopyranoside (1) Yellow amorphous powder, [α]D: 58° (c 0.1, MeOH); UV λmax (MeOH) nm (log ε): 255 (3.15), 320 (3.97); 1H and 13C NMR (400 and 100 MHz, CD3OD): see Table 1; HRESIMS: m/z 917.23510 [M–H]− (calcd for C42H46O23, 918.24299); ESIMS: m/z 917 [M–H]−, 755 [M–H–162]−, 609 [M–H–162–146]−, 301 [M-H-162-146-162-146]−. 2 . 3 . 2 . Quercetin 3-O-β- D - glucopyranoside-7-O-β- D glucopyranosyl-(1→ 3)-α-L-rhamnopyranoside (2) Yellow amorphous powder, [α]D: 45° (c 0.1, MeOH); UV λmax (MeOH) nm (log ε): 256 (3.89), 325 sh (3.51), 360 (3.76); 1H and 13C NMR (400 and 100 MHz, CD3OD): see Table 1; HRESIMS: m/z 771.19868 [M–H]− (calcd for C33H40O21, 772.20621); ESIMS: m/z 771 [M–H]−, 609 [M– H–162]−, 463 [M–H–162–146]−, 301 [M–H–162–146–162]−. 2.3.3. Kaempferol 3-O-β- D -galactopyranoside-7-O-α- L arabinopyranoside (4) Yellow amorphous powder, [α]D: -32° (c 0.1, MeOH); UV λmax (MeOH) nm (log ε): 265 (3.78), 348 (3.71); 1H and 13C NMR (400 and 100 MHz, CD3OD): see Table 1; HRESIMS: m/z 603.12970 [M + Na]+ (calcd for C26H28O15, 580.14282); ESIMS: m/z 579 [M–H]−, 447 [M–H–132]−, 285 [M–H–132– 162]−. 2.3.4. Acid hydrolysis of compounds 1, 2, and 4 A solution of each compound (2.0 mg) in 1 N HCl (1 ml) was stirred at 80 °C in a stoppered reaction vial for 4 h. After cooling, the solution was evaporated under a stream of N2. Each residue was dissolved in 1-(trimethylsilyl)imidazole and pyridine (0.2 ml), and the solution was stirred at 60 °C for 5 min. After drying, the residue was partitioned between H2O and CHCl3. The CHCl3 layer was analyzed by GC using a L-CP-Chirasil-Val column (0.32 mm × 25 m). Temperatures of both the injector and detector were 200 °C. A temperature gradient system was used for the oven, starting at 100 °C for 1 min and increasing up to 180 °C at a rate of 5 °C/min. Peaks of the hydrolysate were detected by comparison with retention times of authentic samples of D-glucose, D-galactose, L-arabinose, and L-rhamnose (Sigma Aldrich), after treatment with 1-(trimethylsilyl)imidazole in pyridine. 2.4. Antioxidant activity 2.4.1. Determination of polyphenolic content Total polyphenols of the extracts were quantified colorimetrically by the Folin-Ciocalteau assay, using gallic acid as standard [23]. Briefly, a suitable aliquot of the sample was combined with 50 μl of Folin-Ciocalteau reagent;3 min, 100 μl of a saturated sodium carbonate solution was added and the final volume (2.5 ml) was reached with distilled water. After 1 h of incubation in the dark at room temperature, the
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absorbance was read at 725 nm and molarity was referred to as gallic acid equivalents. 2.4.2. DPPH scavenging test An appropriate aliquot of the extracts or pure compounds (at concentrations ranging from 1 to 100 μM) was added to a 15 μM ethanol solution of DPPH (2,2-diphenyl-1-picrylhydrazil radical). The samples were incubated in the dark for 15 min and
their absorbance was measured at 517 nm [24]. The assay was performed in triplicate and the IC50 was calculated with Prism® 4 (GraphPad Software Inc.) software. 2.4.3. Total antioxidant capacity The total antioxidant capacity of the samples was determined on the basis of the copper reducing (Cu++ to Cu+) ability by a validated method (BIOXYTECH® AOP-490™, Oxis
Table 1 1 H and 13C NMR data of compounds 1, 2, and 4 (CD3OD, 400 and 100 MHz, J in Hz, δ in ppm) a. 1 δΗ 2 3 4 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' 3-O-Glc 1 2 3 4 5 6a 6b 7-O-Rha 1 2 3 4 5 6 Glc 1 2 3 4 5 6a 6b COO α β 1 2/6 3/5 4 3-O-Gal 1 2 3 4 5 6a 6b 7-O-Ara 1 2 3 4 5a 5b a
6.32 d (2.0) 6.33 d (2.0)
7.72 d (2.0)
6.90 d (8.5) 7.63 dd (8.5, 2.0) 5.32 d (7.5) 3.49 dd (9.0, 7.5) 3.50 t (9.0) 3.33 t (9.0) 3.40 m 3.80 dd (12.0, 3.0) 3.60 dd (12.0, 5.0) 5.41 d (1.8) 4.32 dd (3.0, 1.8) 3.84 dd (9.0, 3.0) 3.65 t (9.0) 3.60 m 1.22 d (6.5) 4.54 d (7.5) 3.42 dd (9.0, 7.5) 3.31 t (9.0) 3.43 t (9.0) 3.68 m 4.54 dd (12.0, 3.0) 4.52 dd (12.0, 5.0) 6.25 d (16.0) 7.54 d (16.0) 7.13 d (8.0) 6.44 d (8.0)
2 δΧ 158.0 135.5 179.4 162.5 100.0 163.5 95.5 156.8 105.0 122.8 117.1 145.8 149.0 115.9 123.3 104.0 75.5 77.9 71.3 78.1 62.5 99.0 71.1 83.4 72.0 70.6 18.0 106.1 75.3 78.0 71.0 75.5 64.8
δΗ
6.49 d (1.8) 6.78 d (1.8)
7.72 d (2.0)
6.85 d (8.5) 7.63 dd (8.5, 2.0) 5.31 d (7.5) 3.45 dd (9.0, 7.5) 3.50 t (9.0) 3.33 t (9.0) 3.38 m 3.80 dd (12.0, 2.5) 3.65 dd (12.0, 4.5) 5.60 d (1.8) 4.32 dd (3.0, 1.8) 3.94 dd (9.0, 3.0) 3.65 t (9.0) 3.66 m 1.28 d (6.5) 4.63 d (7.5) 3.49 dd (9.0, 7.5) 3.38 t (9.0) 3.42 t (9.0) 3.40 m 3.84 dd (12.0, 3.0) 3.60 dd (12.0, 5.0)
4 δΧ 158.2 135.2 179.5 162.5 100.5 163.6 95.5 156.9 105.2 122.5 116.9 146.0 148.7 116.1 123.6 104.3 75.4 77.9 71.2 77.8 62.5
δΗ
6.45 d (1.8) 6.73 d (1.8)
8.12 d (8.0) 6.93 d (8.0) 6.93 d (8.0) 8.12 d (8.0)
δΧ 157.8 135.2 178.3 163.1 98.9 165.4 96.5 159.3 104.5 123.0 132.2 116.2 161.5 116.2 132.2
99.6 71.0 82.5 72.2 70.8 18.0 105.8 75.4 77.7 71.0 77.6 62.5
168.9 114.4 146.8 126.8 131.0 116.2 161.0
Assignments were confirmed by DQF-COSY, 1D-TOCSY, HSQC, and HMBC experiments.
5.58 d (7.8) 3.71 dd (9.0, 7.8) 3.49 dd (9.0, 4.0) 3.65 dd (4.0, 2.5) 3.60 m 3.80 dd (12.0, 2.5) 3.50 dd (12.0, 4.5) 5.02 d (7.0) 3.91 dd (9.0, 7.0) 3.49 dd (9.0, 2.5) 3.70 m 3.90 dd (12.0, 2.0) 3.63 dd (12.0, 3.0)
101.7 72.9 77.5 73.5 77.3 65.3 106.4 72.1 78.0 73.1 65.4
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Research™, Portland, OR) [25]. The samples were tested at concentrations of 10 and 1 μM. The results were shown as mEq uric acid. 3. Results and discussion 3.1. Morphological analysis In order to evaluate the morphologic traits more suitable for the species determination, four taxonomic keys [1–4] have been used. In particular, ten characters were used as diacritic for
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A. burnatii: the roots of the entities appear tuberous; the leaves are divided with linear segments not more than 3 mm wide; the plant stalk is straight; the inflorescence is simple; the flowers are blue-violet; the ratio height/width of helmet is up to 0.7; only glandular hairs along the inflorescence are densely present; the nectaries appear bent forward; the fruits are glabrous follicles; the seeds present three smooth or rugolose sides with three winged angles, one larger than other two. The same number of characters allowed the A. variegatum identification: the roots are tuberous; the leaves are divided to the base and their segments, more than 3 mm wide, are often
Fig. 1. A. burnatii Gáyer.
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Fig. 2. A. variegatum L.
divided to beyond the middle and deeply incise-dentate; the plant stalk has a zigzag line; the inflorescence is branched; the flowers are blue or variegated with helmet about twice as high as wide; only eglandular hairs along the inflorescence are present; the nectaries appear straight; the follicles are hairy at the sutures; the seeds are winged on one angle with prominent transverse lamellae on the sides. Figs. 1 and 2 show the discriminant characters of two species under study, useful for their identification. On the basis of morphological data, the population from Colle di Sampeyre (Cuneo) and Bormio (Sondrio), Italy, can be ascribed to the species Aconitum burnatii and A. variegatum, respectively.
3.2. Phytochemical study The methanol extracts of A. burnatii and A. variegatum aerial parts were chromatographed on Sephadex LH-20 and RP-HPLC (C-18) to yield pure compounds 1–6 (Figs. 3 and 4).
Compound 1, a yellow amorphous solid, showed molecular formula C42H46O23 by means of HRESIMS ([M-H]− peak at m/z 917.23510). Its ESIMS spectrum showed peaks at m/z 755 [M–H–162]−, 609 [M–H–162–146]−, and 301 [M–H– 162–146–162–146]− due to the loss of two hexoses, one deoxyhexoses, and one p-coumaroyl residue. The UV spectrum of 1 showed two absorption maxima at 320 and 255 nm, indicating the presence of substituted aromatic rings and α,β unsaturated ketone in the molecule. The 1H and 13C NMR spectra of compound 1 (Table 1) showed a typical pattern of a flavonol with quercetin aglycon and signals ascribable to sugar moieties and acyl residue [26]. The three anomeric protons arising from the sugar moieties appeared at δ 5.41 (1H, d, J = 1.8 Hz), 5.32 (1H, d, J = 7.5 Hz), and 4.54 (1H, d, J = 7.5 Hz), which correlated respectively with signals at 99.0, 104.0, and 106.1 in the HSQC spectrum. The 1H NMR showed also the presence of a p-coumaroyl residue (Table 1). Assignments of all chemical shifts of protons and carbons of the aglycon, sugar, and acyl portions were ascertained from a
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Fig. 3. Structures of flavonol glycosides 1–3 isolated from A. burnatii.
combination of 1D-TOCSY, DQF-COSY, and HSQC analysis. The configurations of the sugar units were assigned after hydrolysis of 1 with 1 N HCl. The hydrolysate was trimethyl-
silylated and GC retention times were compared with those of authentic sugar samples prepared in the same manner. The lower field shifts of H-6a and H-6b (δ 4.52, 4.54) and C-6
Fig. 4. Structures of flavonol glycosides 4–6 isolated from A. variegatum.
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Table 2 Antioxidant activity of A. burnatii and A. variegatum crude extracts and flavonols. Samples
Polyphenols (mg/g extract)
Ascorbic acid TroloxTM Quercetin
DPPH (IC50) a
Antioxidant capacity (mEq uric acid) b
μM
1 μM
10 μM
9.25 ± 0.89 12.9 ± 0.95 4.37 ± 0.21
0.36 ± 0.02 0.85 ± 0.07 0.75 ± 0.06
0.33 ± 0.02 1.77 ± 0.18 2.17 ± 1.35
A. burnatii extract 1 2 3
173.45 ± 1.58
13.2 ± 0.76 11.8 ± 0.54 12.1 ± 0.55 2.02 ± 0.24
0.37 ± 0.12 0.10 ± 0.09 0.09 ± 0.03 0.15 ± 0.07
1.49 ± 0.65 0.54 ± 0.07 0.46 ± 0.06 0.81 ± 0.11
A. variegatum extract 4 5 6
113.47 ± 1.32
26.7 ± 0.67 356 ± 1.11 295 ± 1.02 277 ± 1. 19
0.12 ± 0.09 0.04 ± 0.02 0.04 ± 0.01 0.05 ± 0.01
1.08 ± 0. 23 0.06 ± 0.01 0.33 ± 0.04 0.46 ± 0.06
a
IC50 = sample concentration required for 50% DPPH free radical scavenging. mEq uric acid = unit of measurement of the antioxidant capacity for copper reduction. Experiments were performed in triplicate and results are mean ± SD. Ascorbic acid, Trolox®, and quercetin are the standard references. b
(64.8) signals of one glucosyl unit, together with the lower field shifts of C-3 signal of rhamnose unit (83.4 ppm), suggested the substitution pattern of glycosyl and acyl moieties. The substitution sites on the molecule were confirmed by an HMBC experiment, showing correlation peaks between δ 5.32 (H-1glcI) and 135.5 (C-3), δ 4.54 (H1glcII) and 83.4 (C-3rha), δ 5.41 (H-1rha) and 163.5 (C-7), δ 4.52 and 4.54 (H-6glcII) and 168.9 (COO). From these results, the structure of 1 was determined to be quercetin 3-O-β-Dglucopyranoside-7-O-(6-E-p-coumaroyl)-β-D-glucopyranosyl-(1 → 3)-α-L-rhamnopyranoside. Compound 2 was obtained as a yellow amorphous powder, with the molecular formula C33H40O21, as deduced from the [MH]− peak at m/z 771.19868 by HRESIMS and confirmed by 13C and 13C DEPT data. The ESIMS of compound 2 showed a prominent fragment at m/z 771 [M-H]− and a fragmentation pattern similar to that of 1. The spectroscopic data of the aglycone moiety of 2 were identical to those of 1. The proton coupling network within each sugar residue was established, using a combination of 1D TOCSY, DQF-COSY, and HSQC experiments. Again, direct evidence for the sugar sequence and the linkage sites was derived from the HSQC and HMBC data. Comparison of NMR data of the sugar moiety (Table 1) of 2 with that of 1 indicated that 2 differed from 1 only by the absence of the p-coumaroyl moiety. The configuration of the sugar units was determined as reported for compound 1. Thus, compound 2 was defined as quercetin 3-O-β-D-glucopyranoside-7-O-β-D-glucopyranosyl-(1 → 3)-α-L-rhamnopyranoside (2). The molecular formula C26H28O15 was assigned to compound 4 as determined by HRESIMS ([M + Na]+ at m/z 603.12970). The UV spectrum was similar to those of 1, suggesting a flavonol glycoside structure. The 1H and 13C NMR of 4 (Table 1) were consistent with a kaempferol glycoside with signals ascribable to aglycon portion and sugar moieties. The two anomeric protons arising from the sugar residues appeared at δ 5.58 (1H, d, J = 7.8 Hz) and 5.02 (1H, d, J = 7.0 Hz) which correlated respectively with signals at 101.7 and 106.4 ppm in the HSQC spectrum. Assignments of all chemical shifts of protons and carbons of the aglycon and sugar
portions were ascertained from a combination of 1D-TOCSY, DQF-COSY, and HSQC analysis. The configurations of the sugar units were assigned after hydrolysis of 4 with 1 N HCl. The hydrolysate was trimethylsilylated and GC retention times were compared with those of authentic sugar samples prepared in the same manner. The substitution sites on the molecule were confirmed by an HMBC experiment showing correlation peaks between δ 5.58 (H-1gal) and 135.5 (C-3), δ 5.02 (H-1ara) and 165.4 (C-7). From these results, the structure of 1 was determined to be kaempferol 3-O-β-D-galactopyranoside-7-Oα-L-arabinopyranoside. Three known flavonols, quercetin 3-O-β-D-glucopyranoside-7-O-(6-E-caffeoyl)-β- D- glucopyranosyl-(1 → 3)-α- Lrhamnopyranoside (3), quercetin 3-O-β-D-glucopyranoside (5), and kaempferol 3-O-β-D-glucopyranoside (6) were also isolated and identified by spectral analysis and comparison of data with those reported in the literature [26,27]. Compound 3 was formerly identified in A. napellus ssp. neomontanum (Wulfer) Gáyer [27], while 5 and 6 were common flavonol glycosides characterized also from A. jaluense complex [7]. 3.3. Antioxidant activity The methanolic extracts obtained from A. burnatii and A. variegatum respectively, were tested for their polyphenolic content by the Folin-Ciocalteau method. The highest concentration of polyphenols was found in A. burnatii (Table 2). The two crude extracts were also analyzed to assess their antioxidant potential by employing different in vitro assays, i.e., DPPH radical scavenging test and total antioxidant capacity based upon the reduction of Cu++ to Cu+. In both assays, ascorbic acid, Trolox®, and quercetin were used as standards. As shown in Table 2, A. burnatii extract displayed a higher activity in comparison with A. variegatum residue. It exhibited a removal action of the stable radical DPPH twice stronger than that of A. variegatum extract. This action was similar to that of ascorbic acid and Trolox®, but three times lower than that of quercetin. Concerning overall antioxidant capacity, activity was noted, for both samples, at concentration of 10 μM (Table 2). From these extracts, compounds 1–6
S. Vitalini et al. / Fitoterapia 81 (2010) 940–947
were isolated and then tested for their antioxidant power (Table 2). The scavenging abilities of 1 and 2 were comparable with those of vitamin C and Trolox®, compound 3 showed a value similar to quercetin, in line with literature data. The catechol structure conferred high stability to the radical form and participated in the electron delocalisation. The caffeoyl derivative 3 had a higher radical scavenging ability than the p-coumaroyl ester 1 [16]. The presence of the caffeoyl moiety enhances the activity of 3 in comparison with 5 [9]. Compounds 4, 5, and 6 were poorly active. All six molecules exhibited limited antioxidant capacity at 1 μM which increased at 10 μM, particularly for 3. Acknowledgements The authors thank Dr. Cristina Mariani for photo presentation and Dr. Marcello Iriti for his cooperation. References [1] Seitz W. Die Taxonomie der Aconitum napellus-Gruppe in Europa. Feddes Repertorium 1969;80:1–76. [2] Hegi G. Illustrierte Flora von Mitteleuropa. München: Carl Hansen; 1974. p. 153–77. [3] Pignatti S. Flora d'Italia, vol. 1. Bologna: Edagricole; 1982. p. 285–8. [4] Akeroyd JR, Charter AO, Aconitum L. In: Tutin TG, Burges NA, Charter AO, Edmondson JR, Heywood VH, Moore DM, Valentie DH, Walters SM, Webb DA, editors. Flora Europaea. 2nd Eds. Cambridge: Cambridge University Press; 1993. p. 254–6. [5] Tutin TG, Burges NA, Charter AO, Edmondson JR, Heywood VH, Moore DM, Valentie DH, Walters SM, Webb DA. Flora Europaea. Cambridge: Cambridge University Press; 1993. [6] Mücher W. Die gattung Aconitum in Kärnten. Carinthia II 1993;183/103: 519–27. [7] Lim CE, Park JK, Park CW. Flavonoid variation of the Aconitum jaluense complex (Ranunculaceae) in Korea. Plant Syst Evol 1999;218:125–31. [8] Fico G, Spada A, Braca A, Agradi E, Morelli I, Tomè F. RAPD analysis and flavonoid composition of Aconitum as an aid for taxonomic discrimination. Bioch Syst Ecol 2003;31:293–301. [9] Mariani C, Braca A, Vitalini S, De Tommasi N, Visioli F, Fico G. Flavonoid characterization and in vitro antioxidant activity of Aconitum anthora L. (Ranunculaceae). Phytochemistry 2008;69:1220–6.
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