Benzophenone O-glycosides from hypericum elegans

Article abstract of DOI:10.1080/14786410802278327

Elegaphenonoside, a new benzophenone O-rhamnoside, together with two known benzophenone O-glycosides, namely hypericophenonoside and neoannulatophenonoside, were isolated from the aerial parts of Hypericum elegans Stephan ex Willd. The structure of the ne

Full text of DOI:10.1080/14786410802278327

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Benzophenone O-glycosides from
Hypericum elegans
Paraskev T. Nedialkov a , Dimitrina Zheleva-Dimitrova a , Ulrich
Girreser b & Gerassim M. Kitanov a
a Department of Pharmacognosy, Faculty of Pharmacy , Medical
University of Sofia , Sofia, Bulgaria
b Institute of Pharmacy, Christian-Albrechts-University of Kiel ,
Kiel, Germany
Published online: 04 Nov 2010.
To cite this article: Paraskev T. Nedialkov , Dimitrina Zheleva-Dimitrova , Ulrich Girreser &
Gerassim M. Kitanov (2009) Benzophenone O-glycosides from Hypericum elegans , Natural Product
Research: Formerly Natural Product Letters, 23:13, 1176-1180, DOI: 10.1080/14786410802278327
To link to this article: http://dx.doi.org/10.1080/14786410802278327
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Natural Product Research
Vol. 23, No. 13, 10 September 2009, 1176–1180
Benzophenone O-glycosides from Hypericum elegans
a
a
b
*
Paraskev T. Nedialkov , Dimitrina Zheleva-Dimitrova , Ulrich Girreser and
Gerassim M. Kitanova
aDepartment of Pharmacognosy, Faculty of Pharmacy, Medical University of Sofia, Sofia,
Bulgaria; Institute of Pharmacy, Christian-Albrechts-University of Kiel, Kiel, Germany
b
(Received 25 May 2008; final version received 14 June 2008)
Elegaphenonoside, a new benzophenone O-rhamnoside, together with two known
benzophenone O-glycosides, namely hypericophenonoside and neoannulatophe-
nonoside, were isolated from the aerial parts of Hypericum elegans Stephan ex
Willd. The structure of the new compound was established as 30,50,6-trihydroxy-4-
methoxybenzophenone-2-O-ꢀ-L-rhamnopyranoside by means of chemical and
physical evidence. In addition, the presence of kaempferol, quercetin, isoquerci-
trin, norathyriol, I-3,II-8-biapigenin, quercitrin, hyperoside and rutin were
established in this plant.
Keywords: Hypericum elegans Stephan ex Willd; elegaphenonoside; benzophe-
none O-glycosides
1. Introduction
Hypericum elegans Stephan ex Willd is a herbaceous plant growing on the Balkan Peninsula,
Mala Asia, Eastern and Middle Europe (Jordanov & Kozˇ ucharov, 1970). No detailed
phytochemical investigation of H. elegans has been undertaken so far. The presence of the
naphthodianthrones hypericin and pseudohypericin (Kitanov, 2001), quercitrin (Kitanov &
Blinova, 1987), and the xanthone C-glucosides mangiferin and isomangiferin (Kitanov &
Nedialkov, 1998) in this species were established by TLC screening. This article is a part of
an extensive investigation of H. elegans and deals with the isolation and structural
elucidation of elegaphenonoside, a new benzophenone O-rhamnoside.
2. Results and discussion
The plant material was extracted with chloroform and then with methanol. The combined
methanolic extract was subjected to a series of chromatographic procedures that led to the
isolation of elegaphenonoside (1), along with two known benzophenone O-glycosides,
as well as seven flavonoids and a xanthone.
25
Elegaphenonoside (1) was isolated as optically active white needles with ½ꢀꢀD ꢁ25.8. Its
molecular formula was established as C20H22O10 by means of FTMS data (m/z 423.1287
[M þ H]þ). The IR spectrum of 1 showed absorption bands of hydroxyls (3356 cmꢁ1),
*Corresponding author. Email: pnedialkov@gmail.com
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2009 Taylor & Francis
DOI: 10.1080/14786410802278327
http://www.informaworld.com

Natural Product Research
1177
chelated carbonyl (1620 cmꢁ1) and aromatic double bonds (1583 cmꢁ1 and 1462 cmꢁ1).
The bathochromic shift of the band at 336 nm in the UV spectrum with AlCl3 (Á ¼ 36 nm)
revealed the presence of a free hydroxyl group in the ortho position to the carbonyl
function. Acid hydrolysis of 1 gave 2,30,50,6-tetrahydroxy-4-methoxybenzophenone
1
(annulatophenone) and L-rhamnose. The signals in the H and 13C spectra were assigned
unambiguously using 2D NMR techniques, i.e. COSY, long-range COSY, NOESY,
HETCOR and COLOC. Multiplicities were revealed by DEPT experiments. The 1H-NMR
spectrum of compound 1 showed two doublets at ꢁH 6.19 and ꢁH 6.38, belonging to the
hydrogens of ring A, as well as a triplet and a doublet at ꢁH 6.46 and ꢁH 6.62, belonging to
the hydrogens of ring B. In addition, the signal of the methoxyl group appeared at ꢁH 3.81.
These showed cross-peaks with the signals at ꢁC 96.1 (C-5), 94.6 (C-3), 107.9 (C-40), 108.2
(C-20 and C-60) and 56.0 (MeO at C-4), respectively. Furthermore, the signals of eight
quaternary carbons, belonging to the aglycone moiety appeared at ꢁC 198.8 (C¼O), 165.4
(C-4), 161.1 (C-6), 159.6 (C-30 and C-50), 158.6 (C-2) 143.3 (C-10) and 110.3 (C-1) in the
1
13C-NMR spectrum of 1, as well. Both H and 13C signal patterns were similar to that of
2-O-glycosides of annulatophenone previously isolated from H. annulatum (Momekov
et al., 2006; Nedialkov & Kitanov, 2002). The relatively small coupling constant
(J ¼ 1.8 Hz) of the doublet at ꢁH 5.26, attributed to the anomeric proton in the sugar
moiety, suggested its ꢀ-configuration. The signals of the other sugar protons appeared as
multiplets at ꢁH 3.40–3.49 (H-200, H-500) and 3.28–3.35 (H-400) as well as a double doublet at
ꢁH 3.15–3.19 (H-300) and a doublet at ꢁH 1.19 (CH3-600). These showed cross-peaks
with signals of six sugar carbons at ꢁC 100.5 (C-100), 73.6 (C-400), 72.0 (C-300), 71.5 (C-200),
70.9 (C-500) and 18.0 (C-600), respectively. The later carbon pattern is typical for
ꢀ-L-rhamnopyranosides (Agrawal, 1992). Computer aided molecular modelling of 1 was
in good agreement with NOESY experiments (Figure 1), where the distances between
˚
correlating protons were 0.23–0.26 nm (2.3–2.6 A). Thus, compound 1 was identified as
30,50,6-trihydroxy-4-methoxybenzophenone-2-O-ꢀ-L-rhamnopyranoside, named elegaphe-
nonoside, which is a new natural product. In addition, hypericophenonoside, neoannu-
latophenonoside, kaempferol, quercetin, isoquercitrin, norathyriol, I-3,II-8-biapigenin,
Figure 1. Selected COLOC and NOESY correlations of 1.

1178
P.T. Nedialkov et al.
quercitrin, hyperoside and rutin were also isolated and identified by the usual techniques.
Benzophenone O-glycosides are rarely encountered in nature. Hypericophenonoside and
neoannulatophenonoside have been previously isolated only from H. annulatum Moris.
(Kitanov & Nedialkov, 2001; Momekov et al., 2006). This article reports the occurrence of
benzophenone O-glycosides in Hypericum species for the second time.
3. Experimental
3.1. General experimental procedures
Melting points (m.p.) were measured on a Kofler hot-stage microscope and are
uncorrected. Optical rotations in MeOH were obtained using a Perkin–Elmer 241
polarimeter. UV spectra were run in MeOH on a Specord UV-VIS instrument; " values are
given in parentheses. IR spectra were recorded with a Shimadzu FTIR-8101 M.
1H and 13C NMR spectra were obtained on a Bruker ARX 300 apparatus at 300 and
75 MHz, respectively, in CD3OD, using tetramethylsilane (TMS) as internal standard.
Electrospray mass spectra (ESIMS) were recorded with a Bruker Esquire-LC ESI-ion trap
in positive mode. High-resolution fourier transform mass spectrum (FTMS) was taken
on a Bruker FT-ICR with electrospray ionisation in methanol containing formic acid.
Column chromatography (CC) was conducted using silica gel 60 (0.063–0.2 mm) and
LiChroprep C-18 (0.04–0.063 mm, an overpressure of 0.8–1.0 bar); flash chromatography
(an overpressure of 0.8–1.0 bar) and vacuum liquid chromatography were performed
with silica gel 60 (0.04–0.063). All adsorbents were purchased from Merck KGaA
(Darmstadt, Germany).
3.2. Collection of biological material
The aerial parts of H. elegans Stephan ex Willd were collected during the Fowering
season from wild habitat at v. Balgarevo, Kavarna in June 2004. A voucher specimen
(No. 153305) was deposited at the Herbarium of the Botany Institute of SoEa (SOM).
3.3. Extraction and isolation
The air-dried and powdered aerial parts (606 g) were refluxed with CHCl3 (6 ꢂ 1 L) at 70ꢃC
for 2 h each time and then extracted with hot MeOH (10 ꢂ 1 L) at 70ꢃC. The crude MeOH
residue (141.30 g) was subjected to CC on silica gel. A stepwise gradient of CH2Cl2
and CH2Cl2–MeOH mixtures (100 : 0 ! 82 : 18, steps of 1%) were used as mobile phase.
A total of 86 fractions (250 mL each) were collected. Based on TLC the identical of these
were combined to give a total of 18 pooled fractions (I–XVIII). Fraction XII was subjected
to gel filtration on Sephadex LH-20, eluted with MeOH–H2O mixtures (50 : 50 ! 100 : 0, in
steps of 10%, 250 mL of each mixture). A total of 50 fractions (30 mL each) were collected.
The fractions 28–30 (80% MeOH) were pooled and subjected to CC on LiChroprep C-18
using MeOH–H2O mixtures (25 : 75 ! 40 : 60, in steps of 1%, 250 mL of each mixture) as
mobile phase. Gel filtration over Sephadex LH-20 (eluent MeOH) was used as a final
purification step, which gave 723.9 mg of compound 1. Similar chromatographic
procedures of fractions XIII–XV gave pure hypericophenonoside and neoannulatopheno-
noside. In addition, kaempferol, quercetin, isoquercitrin, norathyriol, I-3,II-8-biapigenin,
quercitrin, hyperoside and rutin were also isolated by the usual techniques.

Natural Product Research
1179
3.4. Acid hydrolysis of compound 1
A solution of compound 1 (10.0 mg) in 1N HCl (2 mL) was stirred at 100ꢃC under reflux in
a reaction flask for 4 h. Reaction mixture was transferred to separating funnel, diluted to
10 mL with water and extracted with water saturated EtOAc (3 ꢂ 5 mL). Both layers were
separately evaporated to dryness in vacuo. The EtOAc residue was subjected to gel
filtration on Sephadex LH-20 and eluted with MeOH. The aglycone of 1 was obtained as
pale yellow powder and was identified as 2,30,50,6-tetrahydroxy-4-methoxybenzophenone
on the basis of the melting point, co-TLC with an authentic sample, UV, IR and
ESIMS (Kitanov & Nedialkov, 2001). The sugar was identified as L-rhamnose by means of
co-TLC with an authentic sample on cellulose (Merck), mobile phase EtOAc-pyridine-
H2O (12 : 5 : 4) and spots were visualised with anisidine phtalate reagent (heating to 110ꢃC
for 3–5 min).
3.5. Molecular modelling
The optimisation of molecular geometry of 1 was done by ab initio calculation mode, using
GAMESS-US (Schmidt et al., 1993), Version 24 March 2007 R6, on a 32 bit Linux 2.6
box. The parameters were set to as follows: self-consistent field wavefunction was set to
Restricted Hatree Fock (RHF), the initial molecular geometry was given as Gaussian style
internals, Gaussian basis was set to Pople’s STO-NG minimal basis set, the number of
Gaussians was set to 3, C1 was chosen as a symmetry group, all other parameters were set
to defaults. The Molden program package Version 4.6 (Schaftenaar & Noordik, 2000) was
used for drawing initial geometry coordinates as well as for visualising calculation results
and for measuring intra molecular distances.
3.5.1. Elegaphenonoside (1)
25
White needles: m.p. 221–223ꢃC; ½ꢀꢀD ꢁ25.8 (c ¼ 2.01, MeOH); IR (nujol) ꢂmax: 3356.0,
2924.4, 2953.4, 2855.0, 1620.4, 1462.2, 1583.7; UV (MeOH) ꢃmax: 300 (4.25); (þAlCl3):
336, 371sh; (þNaOAc): 285, 371sh; 1H-NMR (300 MHz, CD3OD): 6.62 (2H, d, J ¼ 2.3 Hz,
H-20 and H-60), 6.46 (1H, t, J ¼ 2.3 Hz, H-40), 6.38 (1H, d, J ¼ 2.2 Hz, H-3), 6.19 (1H, d,
J ¼ 2.2 Hz, H-5), 5.26 (1H, d, J ¼ 1.8 Hz, H-100), 3.81 (3H, s, CH3O-4), 3.40–3.49 (2H, m,
H-200 and H-500), 3.28–3.35 (1H, m, H-400), 3.17 (1H, dd, J ¼ 3.4, 9.4 Hz, H-300), 1.20 (3H, d,
J ¼ 6.1 Hz, C-6); 13C-NMR (75 MHz, CD3OD): 198.8 (C¼O), 165.4 (C-4), 161.1 (C-6),
159.5 (C-30 and C-50), 158.5 (C-6), 143.3 (C-10), 110.3 (C-1), 108.2 (C-20 and C-60), 107.9
(C-40), 100.5 (C-100), 96.1 (C-5), 94.6 (C-3), 73.6 (C-400)þ, 72.0 (C-300), 71.þ5 (C-200), 70.9 (þC-500),
56.0 (CH3O-4), 18.0 (C-600); ESIMS: m/z 461 [M þ K] , 445 [M þ Na] , 423 [M þ H] , 405
[M ꢁ H2O þ H]þ, 277 [Magl]þ; FTMS: Calculated for [C20H22O10 þ H]þ 423.1286, found
423.1287; calculated for [C20H22O10 þ Na]þ 445.1105, found 445.1105; calculated for
[(C20H22O10)2 þ Na]þ 867.2318, found 867.2317.
Acknowledgement
This work was supported by Grant 31/2007 from the Medical Science Council at the Medical
University of Sofia.

1180
P.T. Nedialkov et al.
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