Biochemical Systematics and Ecology 33 (2005) 201–205

www.elsevier.com/locate/biochemsyseco

Flavonol 3,7-glycosides anddihydroxyphenethyl glycosides fromAconitum napellus subsp. lusitanicum

Jesus G. Dıaza, Juan Garcıa Ruiza, Bianca Rachid Dıasa,Jose A. Gavın Sazatornila, Werner Herzb,*

aInstituto de Bio-Organica ‘‘A. Gonzalez’’, Universidad de La Laguna,

Ctra a la Esperanza 2, 38206 La Laguna, Tenerife, SpainbDepartment of Chemistry and Biochemistry, The Florida State University, Tallahassee,

FL 32306-4390, USA

Received 14 April 2004; accepted 9 July 2004

Keywords: Aconitum napellus ssp. lusitanicum; Ranunculaceae; Flavonol glycosides

1. Subject and source

Aerial parts of Aconitum napellus L. ssp. lusitanicum Rory were collected onSeptember 18, 1997 in San Emilio, Leon Province, Spain. The plant was identified byProfessor Julian Molero, Department of Botany, Faculty of Pharmacy, Universi-dade de Barcelona. A voucher specimen (No. BCF 43708) is on deposit in theherbarium of that department.

* Corresponding author. Tel.: C1 850 644 2774; fax: C1 850 644 8281.

E-mail address: jdulin@chem.fsu.edu (W. Herz).

0305-1978/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.bse.2004.07.005

202 J.G. Dıaz et al. / Biochemical Systematics and Ecology 33 (2005) 201–205

2. Previous work

Numerous studies have dealt with the diterpene alkaloids of Aconitum species(Ranunculaceae) but reports on other constituents are sparse. An early study dealtwith the phenolic constituents of A. variegatum and A. napellus ssp. skevisorae(Sweykowski and Krzakowa, 1977a,b) and during the last decade articles haveappeared on the flavonoids of several Korean Aconitum species (Whang et al., 1994;Kim et al., 1996; Dae et al., 1996; Jeong et al., 1997). Lim et al. (1999) studiedflavonoid variations in members of the Korean Aconitum jaluense complex; sub-sequently Fico et al. (2001a,b) reported on flavone glycosides from the flowers of twoother subspecies of A. napellus ssp. tauricum and ssp. neomontanum. The same group(Fico et al., 2003) very recently demonstrated that their flavonoid glycoside profilescharacterize the two A. napellus subspecies studied by them earlier and that thesediffered in turn from the flavonoid profiles of A. paniculatum and A. vulparia. Asa result they suggested that species recognition within this large genus might betackled by using flavonoids as chemical molecular markers.

3. Present work

3.1. General procedures

IR spectra were determined using a Bruker IFS-55 spectrometer. 1H and 13CNMR spectra were measured using a Bruker AMX-400 or Bruker MAX-500instruments. EIMS and exact mass measurements were determined using a Micro-mass Autospec instrument at 70 eV. Al2O3 Merck (neutral, 200–300 mesh) andSchleicher and Schuell 394 732 were used for column (CC) and thin layer (TLC)chromatography, respectively. Sephadex was LH-20, Pharmacia (ref. 17-0090-01).HPLC separations were performed on a JASCO Pu-980 series pumping systemequipped with a JASCO UV-975 ultraviolet detector and with a Waters Kromasil�

Si 5 mm (Wx 250 mm) column; flow rate of mobile phase 3 ml�1 with EtOAc–hexane.

3.2. Extraction and isolation of constituents

Air-dried powdered aerial parts (2.56 kg) of A. napellus ssp. lusitanicus wereextracted with 90% EtOH at room temperature for 8 days. Filtration and removal ofsolvent at reduced pressure afforded 232 g of crude extract which was adsorbed on550 g of Si gel and submitted to flash chromatography using hexane (10 l), hexane–EtOAc (1:1, 10 l), EtOAc (10 l), EtOAc–MeOH (3:1, 10 l), EtOAc–MeOH (1:1, 10 l)and MeOH (10 l) to furnish 30 g, 26 g, 141 g, 37.5 g and 92 g of residues in therespective eluates. A portion (15 g) of the EtOAc–MeOH (3:1) fraction werechromatographed on Sephadex LH-20 using MeOH–CHCl3–hexane (1:1:9) as eluentto give 58 fractions of 100 ml each. These were combined into 12 fractions on thebasis of TLC analyses (Si 60 F254 gel coated plastic sheets, eluent EtOAc–MeOH1:2). Fractions 3–4 (75 mg) were combined and acylated with Ac2O–pyridine in the

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usual fashion at room temperature overnight. Removal of the solvents at reducedpressure followed by purification over Sephadex LH-20 (hexane–CHCl3–MeOH,2:1:1) afforded 10 mg of hexaacetate 4b and 12 mg of hexaacetate 5b. Fractions 11–12 (500 mg) were combined and acetylated with Ac2O–pyridine in the usual fashionat room temperature overnight. After the usual work-up flash chromatography andHPLC of the acetylated material gave 1b (RtZ 26 min, 4.5 mg), 2b (RtZ 37 min,2 mg) and 3b (RtZ 43 min, 219 mg).

The five glycoside constituents of A. napellus ssp. lusitanicus, isolated as describedin the preceding paragraph, i.e. quercetin 3-O-b-glucopyranoside-7-O-a-rhamnopyr-anoside (1a), quercetin 3-O-(6-trans-caffeoyl)-b-glucopyranosyl-(1/ 2)-b-glucopyr-anosyl-7-O-a-rhamnopyranoside (2a), quercetin 3-O-(6-trans-p-coumaroyl)-b-glucopyranosyl-(1/ 2)-b-glucopyranosyl-7-O-a-rhamnopyranoside (3a), b-3,4-di-hydroxyphenethyl-b-glucopyranoside (4a) and b-3,4-dihydroxyphenethyl-(6-O-fer-uloyl)-b-glucopyranoside (5a) were identified as their peracetates 1b–5b by means of1H, 13C NMR, COSY and HMBC. The peracetate of 1a (vingetoxicoside A),a common plant constituent, was first reported by Plouvier (1970), but its 1Hspectrum has apparently not been recorded previously and is listed below.Glycosides 2a and 3a were first found in the extract of Aconitum napellus ssp.tauricum flowers (Fico et al., 2001a); glycoside 4a has also been isolated from theflowers of A. napellus ssp. neomontanum (Fico et al., 2001b). 1H and 13C NMRspectra of the previously unreported peracetate of 2a and the 1H NMR spectrum of3b are also listed below. Glycoside 5a (osmanthoside E) was first reported fromPrunus grayana (Shimomura et al., 1987) and subsequently from Osmanthus asiaticusas osmanthuside E by Sugiyamo and Kikuchi (1990) who also reported 1H and 13CNMR spectra of the peracetate.

Quercetin 3-O-glucopyranoside-7-O-a-rhamnopyranoside decaacetate (1b).Amorphous, 1H NMR (400 MHz, CDCl3) d 6.78 (d, JZ 2.5 Hz, H-6,) 7.11 (d,JZ 2.5 Hz, H-8), 7.91 (d, JZ 2 Hz, H-2#), 7.32 (d, JZ 8.5 Hz, H-5#), 7.96 (dd,JZ 8.5, 2 Hz, H-6#), 5.63 (d, JZ 7.8 Hz, H-1–Glc), 5.17 (t, JZ 9.5 Hz, H-2–Glc),5.28 (t, JZ 9 Hz, H-3–Glc), 5.05 (t, JZ 9.7 Hz, H-4–Glc), 3.60 (br d, JZ 8.5 Hz, H-5–Glc), 4.02 (dd, JZ 12.3, 4.2 Hz, H-6a–Glc), 3.94 (dd, JZ 12.3, 2.4 Hz, H-6b–Glc), 5.57 (br d, JZ 1.8 Hz, H-1–Rha), 3.91 (m, H-5 Rha), 1.22 (d, JZ 6.1 Hz, 3H,H-6-Rha), 2.44 s, 2.35 s, 2.32 s, 2.20 s, 2.04 sa, 2.06 s, 1.99 s, 2.03 s, 2.10 s, 1.91 s(each 3H, 5-Ac, 3#-Ac, 4#-Ac, 2-Rha-Ac, 3-Rha-Ac, 4-Rha-Ac, 2-61c-Ac, 3-Glc-Ac,4-Glc-Ac, 6-Glc-Ac).

Quercetin 3-O-(6-trans-caffeoyl)-b-glucpyranosyl-(1/ 2)-b-glucopyranoside-7-O-a-rhamnopyranoside tetradecaacetate (2b). Amorphous, 1H NMR (500 MHz,CDCl3) d 6.71 (d, JZ 2.5 Hz, H-6), 6.94 (d, JZ 2.5 Hz, H-8), 7.92 (d, JZ 2.0 Hz,H-2#), 7.43 (d, JZ 8.5 Hz, H-5#), 7.73 (dd, JZ 8.5, 2.0 Hz, H-6#), 5.90(d, JZ 7.8 Hz), 3-O-Glc-1), 3.98 (dd, JZ 9.2, 7.5 Hz, 3-O-Glc-2), 5.15 (t,JZ 8.9 Hz, 3-O-Glc-3), 5.11 (t, JZ 9.6 Hz, 3-O-Glc-4), 3.67 (m, 3-O-Glc-5), 4.12(dd, JZ 12.3, 4.3 Hz, 3-O-Glc-6a), 3.82 (dd, JZ 12.3, 2.4 Hz, 3-O-Glc-6b), 4.83 (d,JZ 7.8 Hz, 1-Glc2), 5.00 (t, JZ 9.6 Hz, 2-Glc2), 5.04 (t, JZ 8.3 Hz, 3-Glc2), 5.26 (t,JZ 9.5 Hz, 4-Glc2), 3.67 (m, 5-Glc2), 4.07 (dd, JZ 12.3, 4.2 Hz, 6a-Glc2), 3.96 (dd,JZ 12.3, 2.2 Hz, 6b-Glc2), 5.50 (br d, JZ 1.2 Hz, 1-Rha), 5.44 (dd, JZ 3.4, 1.2 Hz,

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2-Rha), 5.41 (dd, JZ 9.6, 3.42 Hz, 3-Rha), 5.43 (d, JZ 10 Hz, 4-Rha), 3.85 (dq,JZ 9.5, 6.2 Hz, 5-Rha), 1.17 (d, JZ 6.2, 3p, Hz, 6-Rha), 6.24 (d, JZ 16 Hz, H-a ofcaff), 7.13 (d, JZ 16 Hz, H-b of caff), 7.21 (d, 2 Hz), 7.07 (d, 8.2 Hz), 7.20 (dd, 8.2,2.0 H, H2#, 5#, H-6# of caff), 2.45 s, 2.36 s, 2.30 s, 2.27 s, 2.25 s, 2.19 s, 2.01 s, 2.04 s,1.96 s, 1.95 s, 1.98 s, 2.01 s, 2.09 s, 1.90 s (all 3H, 5-Ac, 3#-Ac, 4#-Ac, 3-caff-Ac, 4-caff-Ac, 2-Rha-Ac, 3-Rha-Ac, 4-Rha Ac, 3-Glc1Ac, 4-Glc1-Ac, 2-Glc2-Ac, 4-Glc2-Ac, 6-Glc2-Ac, 13C NMR (100 MHz, CDCl3) d 153.7 (C-2), 136.1 (C-3), 172.1 (C-4),150.5 (C-5), 109.7 (C-6), 159.0 (C-7), 101.4 (C-8), 157.3 (C-9), 112.7 (C-10), 128.9 (C-1#), 124.9 (C-2#), 141.7 (C-3#), 143.7 (C-4#), 123.2 (C-5#), 126.8 (C-6#), 97.9 (C-1,Glc1), 77.8 (C-3, Glc1), 77.3 (C-2, Glc1), 68.1 (C-4, Glc1), 71.5 (C-5, Glc1), 61.4 (C-6,Glc1), 100.3 (C-1, Glc2), 68.3 (C-4, Glc2), 71.6 (C-3, Glc2), 74.1 (C-2, Glc2) 71.5 (C-5,Glc2), 61.4 (C-6, Glc2), 95.6 (C-1, Rha), 68.9 (C-2, Rha), 68.7 (C-3, Rha), 70.4 (C-4,Rha), 67.8 (C-5, Rha), 17.4 (C-6, Rha), 165.8 (CO of caff), 118.5 (Ca), 143.0 (Cb),132.9 (C-1#) 122.5 (C-2#), 142.3 (C-3#) 143.5 (C-4#), 123.7 (C-5#), 126.5 (C-6#).Acetates d 170.4, 170.39, 169.9, 169.89, 169.89, 169.8, 169.7, 169.4, 169.3, 169.1,168.1, 168.0, 167.9, 167.7, 21.07, 20.77, 20.77, 20.69, 20.64, 20.60, 20.60, 20.56, 20.54,20.51, 20.51, 20.44, 20.14, 20.37.

Quercetin 3-O-(6-trans-p-coumaroyl)-b-glucopyranosyl-(1/ 2)-b-glucopyrano-side-7-O-a-rhamnopyranoside trisdecaacetate (3b). Amorphous, 1H NMR(400 MHz, CDCl3) d 6.71 (d, JZ 2.5 Hz, H-6), 6.93 (d, JZ 2.5 Hz, H-8), 7.92(d, JZ 2.0 Hz, H-2#), 7.31 (d, JZ 8.5 Hz, H-5#), 7.74 (dd, JZ 8.5, 2.0 Hz, H-6#),5.92 (d, JZ 7.8 Hz, 3-O-Glc-1), 3.86 (dd, JZ 9.2, 6.5 Hz, 3-O-Glc-2), 5.12 (t,JZ 8.9 Hz, 3-O-Glc-3), 5.26 (t, JZ 9.5 Hz, 3-O-Glc-4), 3.69 (m, 3-O-Glc-5), 4.02(dd, JZ 12.5, 4.2 Hz, 3-O-Glc-6a), 3.80 (dd, JZ 12.5, 2.4 Hz, 3-O-Glc-6b), 4.82 (d,JZ 7.8 Hz, 1-Glc2), 5.01 (t, JZ 9.5 Hz, 2-Glc2), 5.05 (t, JZ 8.3 Hz, 3-Glc2), 5.27 (t,JZ 9.5 Hz, 4-Glc2), 3.68 (m, 5-Glc2), 4.14 (dd, JZ 12.3, 4.0 Hz, 6a-Glc2), 3.97 (m,6b-Glc2), 5.45 (br d, JZ 1.2 Hz, 1-Rha), 5.44 (m, 2-Rha), 5.42 (dd, JZ 9.5, 3.4 Hz,3-Rha), 5.14 (t, JZ 10 Hz, 4-Rha), 3.88 (m, 5-Rha), 1.16 (dd, JZ 6.2 Hz, 3H, 6-Rha), 6.27 (d, JZ 16 Hz, H-a of coum), 7.49 (d, JZ 16 Hz, H-b of coum), 6.98 (2H,d, JZ 8.5 Hz), 7.37 (2H, d, JZ 8.5 Hz, H-2#, H-3#, 5# of coum), 2.45 s, 2.37 s, 2.31s, 2.29 s, 2.20 s,2.02 s, 2.05 s, 1.96 s, 1.97 s, 1.99 s, 2.03 sa, 2.10 s, 1.90 s (each 3H, 5#-Ac, 3#-Ac, 4#-Ac, 4-coum-Ac, 3-Rha-Ac, 4-Rha-Ac, 3#-Glc1-Ac, 4-Glc1-Ac, 3-Glc2-Ac, 4-Glc2-Ac, 6-Glc2-Ac).

4. Chemotaxonomic significance

The flavonoid profile of A. napellus ssp. lusitanicum is somewhat similar to that ofssp. tauricum (Fico et al., 2003), although the latter contained in addition to thequercetin derivatives 2a and 3a the corresponding kaempferol analogs which wereabsent from our collection of ssp. lusitanicum. On the other hand the flavonoidprofile of ssp. lusitanicum differ significantly from that of ssp. neomantanum (Ficoet al., 2003). In the latter the glucose unit on C-3 of quercetin or kaempferol isunsubstituted while the rhamnose unit on C-7 contains the 6#-caffeoylglucosyl orcoumaroyl glycosyl-(1/ 2)-glucose combination. However, glycosides 3a, 4a and 5a

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found by us in ssp. lusitanicum were not reported as constituents of either ssp.tauricum or ssp. neomontanum. Our results thus seem to support the contention ofthe Italian workers that flavonoid profiles are useful chemical molecular markerswithin the genus Aconitum.

Acknowledgment

We thank the Instituto Canario de Investigacion del Cancer (CIC) for financialsupport. JGR is indebted to the AECI for a fellowship.

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