Benzopyranones and Their Sulfate Esters from Pelargonium sidoides

Article abstract of DOI:10.1055/s-0029-1186167

Investigation of the benzopyranones from the roots of Pelargonium sidoides led to the identification of 15 benzopyranones, among them the novel compounds 12 and 14. Furthermore, benzopyranones 4 and 15 were detected in Pelargonium sidoides for the first time. Structure determinations were performed by one- and two-dimensional NMR spectroscopy. An HPLC system for the discrimination of these benzopyranones has been developed. Georg Thieme Verlag KG Stuttgart New York.

Full text of DOI:10.1055/s-0029-1186167

350 Original Papers
Benzopyranones and Their Sulfate Esters from
Pelargonium sidoides
Authors
Hermann Hauer1, Stefan Germer2, Jens Elsäßer1, Thomas Ritter1
1
Affiliations
Preclinical Research, Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany
Analytical Development, Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany
2
Key words
Abstract
were performed by one- and two-dimensional
"
l Pelargonium sidoides
!
NMR spectroscopy. An HPLC system for the dis-
crimination of these benzopyranones has been
developed.
"
l Geraniaceae
Investigation of the benzopyranones from the
roots of Pelargonium sidoides led to the identifica-
tion of 15 benzopyranones, among them the nov-
el compounds 12 and 14. Furthermore, benzopyr-
anones 4 and 15 were detected in Pelargonium si-
doides for the first time. Structure determinations
"
l benzopyranones
"
l sulfate esters
"
l HPLC
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
"
l isolation
Introduction
!
Materials and Methods
!
According to a recently published review by
Brendler and van Wyk, Pelargonium reniforme
Curt. and Pelargonium sidoides DC. (Geraniaceae)
were used traditionally to treat diseases like dys-
entery and diarrhea, but not respiratory tract in-
fections [1]. In 1897, this drug was introduced to
England by Stevens [2,3]. In modern phytother-
apy, an extract preparation from the roots of Pe-
largonium sidoides, Umckaloabo® [4], is used
against infections of the respiratory tract.
HPLC fingerprint
HPLC analysis was performed on a reversed phase
(C18) column (Phenomenex; Synergi Hydro-RP,
4 µm, 250 × 3 mm) with a column temperature of
60°C and linear gradient elution with mobile
phase A (acetonitrile:water = 1:99 v/v + 3 mL or-
thophosphoric acid/L) and mobile phase B (aceto-
nitrile:water = 40:60 v/v + 3 mL orthophosphoric
acid/L) from 100% A to 50% A in 47 min, flow rate
1.0 mL/min and detection at UV 330 nm.
Benzopyranone derivatives are present in an
amount of about 0.2% in the roots of Pelargonium
sidoides. Several benzopyranones have been iso-
lated and described up to now [5–7]. Among
these compounds, 7-hydroxy-5,6-dimethoxy-
2H-1-benzopyran-2-one, “umckalin” [2,3], was
assumed to be the principal benzopyranone [5].
HPLC analysis of extracts from Pelargonium si-
doides showed several other peaks, which were
identified by isolation and structure elucidation
of the corresponding compounds. All these com-
pounds were identified as highly oxygenated
benzopyranones, some of them were described
for the first time.
NMR spectroscopy
received
revised
accepted
June 19, 2009
August 6, 2009
August 27, 2009
1H- and 13C‑NMR spectra were acquired on a
Bruker DPX200 spectrometer (200 MHz) in
DMSO-d6. The chemical shifts refer to TMS
Bibliography
(1H‑NMR) or to DMSO-d6
39.5 ppm), respectively.
(
13C‑NMR, δ =
DOI 10.1055/s-0029-1186167
Published online October 6,
2009
Planta Med 2010; 76: 350–352
© Georg Thieme Verlag KG
Stuttgart · New York ·
ISSN 0032‑0943
Mass spectroscopy
ESI‑MS data were obtained by direct infusion of
solutions on a Bruker Esquire HCT ion trap mass
spectrometer. Sample solution: about 0.1 mg/mL
in acetonitrile:water = 1:1 v/v + 0.1% formic acid.
Detected were negative ions, which were sequen-
tially fragmented.
Correspondence
Dr. Hermann Hauer
Preclinical Research
Dr. Willmar Schwabe GmbH &
Co. KG
PO Box 410925
76209 Karlsruhe
Germany
Phone: + 497214005387
Fax: + 4972140058387
hermann.hauer@schwabe.de
Isolation and preparation
Isolation and 1H- and 13C‑NMR data of the com-
pounds 1, 2 and 5 to 10 as well as the plant mate-
rial used are described in ref. [6].
Hauer H et al. Benzopyranones and Their… Planta Med 2010; 76: 350–352

Original Papers 351
For the isolation of compounds 4, 12, 14 and 15, dried roots of
Pelargonium sidoides (15 kg) were extracted with water (115 L).
The extract was concentrated to a weight of 35 kg, saturated with
ammonium sulfate (7 kg) and extracted three times with a mix-
ture of 2-butanone and ethanol (3:2; 40 L in total). The organic
phase was concentrated to 890 g, dissolved in water (10 L) and
chromatographed on Diaion HP-20 [20 × 32 cm; eluent: water
(30 L) → 10% ethanol (30 L)]. For further separation, the fractions
were treated as described below. All pH adjustments mentioned
below were performed using diluted potassium hydroxide solu-
tion. Therefore, all sulfooxy residues were deprotonated to yield
the corresponding potassium salts.
144.9 (CH, C-4), 143.2 (C, C-6), 142.8 (C, C-7), 139.5 (C, C-8a),
137.8 (C, C-8), 114.2 (CH, C-3), 113.8 (C, C-4a), 109.8 (CH, C-5),
60.7 (CH3, OCH3); ESI‑MS (neg. ion mode): m/z =
287.0 → 206.5 → 191.6.
Conversion of 14 to 6,8-dihydroxy-7-methoxy-2H‑1-benzopyran-
2-one (13): Compound 14 (60 mg) was heated for 30 min in 2 M
HCl. Compound 13 precipitated on cooling and was washed with
water (yield: 30 mg, 78%). 1H‑NMR data of 13 correspond to
those published by Kayser [9]. Furthermore, 1H- and 13C‑NMR da-
ta in DMSO-d6 were in agreement with those of ref. [6] (see Table
1S, Supporting Information).
6,8-Bis(sulfooxy)-7-methoxy-2H‑1-benzopyran-2-one (15): Isola-
tion: The fractions containing 15 were adjusted to pH 7.5 and
concentrated. Purification was performed by column chromatog-
raphy on Sephadex LH-20 (5.5 × 100 cm; methanol) and RP-18
(2.6 × 46 cm; water/acetonitrile, 99:1), adjustment to pH 7.5 and
washing with methanol yielding the dipotassium salt of 15
(yield: 0.18 g). Synthesis: Compound 15 was prepared by alkyla-
tion of 10 (2.0 g) with methyl iodide (11.2 g) in dimethylforma-
mide (80 mL) in the presence of potassium carbonate (10.7 g).
The reaction mixture was evaporated and the residue diluted
with water, adjusted to pH 6.5 with HCl and filtered. Purification
of 15 was performed by column chromatography on Diaion HP-
20 (5 × 22 cm; water), adjustment to pH 8, evaporation and crys-
tallization with methanol yielding the dipotassium salt of 15
7-Hydroxy-6-methoxy-2H‑1-benzopyran-2-one (scopoletin, 3):
Compound 3 was prepared as described by Hauer et al. [8]. 1H-
and 13C‑NMR see Table 1S (Supporting Information).
6-Methoxy-7-(sulfooxy)-2H‑1-benzopyran-2-one (4): Isolation:
The fractions containing 4 were adjusted to pH 7.5 and concen-
trated (8 g). Purification was performed by column chromatogra-
phy on Sephadex LH-20 (5.5 × 100 cm; methanol) and Lichroprep
RP-18 (40–63 µm, 2.6 × 46 cm; water/acetonitrile 99:1 → 97:3,
10 mL/min, 15 bar), adjustment to pH 7.5 and evaporation yield-
ing the monopotassium salt of 4 (0.12 g). Synthesis: Compound 3
(5.0 g), SO3-NMe3 (4.2 g) and K2CO3 (4.0 g) were stirred in DMF
(20 mL) at room temperature for 18 h under N2. The mixture
was evaporated and the residue was recrystallized from water
and from ethanol/water 9/1 affording the monopotassium salt of
4 (yield: 3.0 g, 37%). 1H‑NMR (DMSO-d6, 200 MHz): δ = 7.97 (1H,
d, J = 9.5 Hz, H-4), 7.51 (1H, s, H-8), 7.29 (1H, s, H-5), 6.35 (1H, d,
J = 9.5 Hz, H-3), 3.80 (3H, s, OCH3); 13C NMR (DMSO-d6, 50 MHz):
δ = 160.4 (C, C-2), 148.1 (C, C-8a), 147.0 (C, C-6), 146.3 (C, C-7),
144.2 (CH, C-4), 113.7 (CH, C-3), 113.2 (C, C-4a), 109.7 (CH, C-5),
107.2 (CH, C-8), 56.0 (CH3, OCH3).
1
(yield: 0.9 g). H- and 13C‑NMR data in DMSO-d6 were in agree-
ment with those of ref. [6] (see Table 1S, Supporting Informa-
tion).
Supporting information
1H- and 13C‑NMR data of the benzopyranones 1 to 15 are avail-
able as Supporting Information.
7-Hydroxy-6-methoxy-8-(sulfooxy)-2H‑1-benzopyran-2-one (12):
The fractions containing 12 were adjusted to pH 7.5, concen-
trated (14 g) and purified by column chromatography on Sepha-
dex LH-20 (5.5 × 100 cm; methanol) and washed with methanol,
affording the monopotassium salt of 12 (yield: 0.45 g). 1H- and
13C‑NMR see Table 1S (Supporting Information). 1H‑NMR
(DMSO-d6, 200 MHz): δ = 9.73 (1H, s, OH), 7.94 (1H, d, J = 9.4 Hz,
H-4), 7.10 (1H, s, H-5), 6.28 (1H, d, J = 9.4 Hz, H-3), 3.81 (3H, s,
OCH3); 13C NMR (DMSO-d6, 50 MHz): δ = 160.2 (C, C-2), 146.0 (C,
C-6), 144.6 (CH, C-4), 144.3 (C, C-7), 142.9 (C, C-8a), 128.3 (C, C-8),
112.5 (CH, C-3), 110.6 (C, C-4a), 105.8 (CH, C-5), 56.0 (CH3, OCH3);
ESI‑MS (neg. ion mode): m/z = 287.0 → 207.1 → 192.1.
Results and Discussion
!
"
The HPLC fingerprint shown in l Fig. 1 was obtained with an
aqueous ethanolic extract (11 weight % ethanol in water) of dried
roots from Pelargonium sidoides. The peaks of the benzopyra-
"
"
nones 1 to 15 (l Fig. 2 and l Table 1) in the HPLC fingerprint
were identified by comparison with the retention times and
DAD spectra of the reference substances 1 to 15, which were iso-
lated from Pelargonium sidoides or synthesized and identified by
NMR spectroscopy. Peak identification was furthermore verified
Conversion of 12 to 7,8-dihydroxy-6-methoxy-2H‑1-benzopyran-
2-one (fraxetin, 11): Compound 12 (52 mg) was heated for
20 min in 2 M HCl. Compound 11 precipitated on cooling and
was washed with water (yield: 20 mg, 60%). 1H- and 13C‑NMR da-
ta in DMSO-d6 were in agreement with those of a commercially
available sample (Extrasynthèse). 1H- and 13C‑NMR see Table 1S
(Supporting Information).
8-Hydroxy-7-methoxy-6-(sulfooxy)-2H‑1-benzopyran-2-one (14):
The fractions containing 14 were adjusted to pH 7.5 and concen-
trated (8 g). Purification was performed by column chromatogra-
phy on Sephadex LH-20 (5.5 × 100 cm; methanol) and Lichroprep
RP-18 (40–63 µm, 2.6 × 46 cm; water/acetonitrile 99:1 → 97:3,
10 mL/min, 15 bar), adjustment to pH 7.5 and washing with
methanol affording the monopotassium salt of 14 (yield: 0.58 g).
1H- and 13C‑NMR see Table 1S (Supporting Information). 1H‑NMR
(DMSO-d6, 200 MHz): δ = 8.25 (1H, br. s, OH), 7.98 (1H, d,
J = 9.5 Hz, H-4), 7.27 (1H, s, H-5), 6.33 (1H, d, J = 9.5 Hz, H-3), 3.85
(3H, s, OCH3); 13C‑NMR (DMSO-d6, 50 MHz): δ = 160.1 (C, C-2),
Fig. 1 HPLC fingerprint of Pelargonium sidoides.
Hauer H et al. Benzopyranones and Their… Planta Med 2010; 76: 350–352

352 Original Papers
sponding spectra of commercially available fraxetin. The 1H
-NMR spectrum of 13 corresponds to the spectrum published by
Kayser [9].
Fig. 2 Chemical struc-
tures of benzopyra-
nones 1 to 15.
NMR assignments were performed by two dimensional methods
(NOESY, HMBC). NMR shifts and 1H‑NMR coupling constants are
given in Table 1S (Supporting Information).
NOESY spectra of the compounds 7 to 14 gave strong correlations
between H-4 and H-5 showing trisubstitution in the positions 6,
7, and 8. Additionally, H-5/6-OCH3 correlations in compounds 11
and 12 indicated the 6-position of the OCH3 group. An HMBC cor-
relation of C-7 with the OCH3 protons of 13 confirmed the me-
thoxylation in position 7.
The substitution patterns of the sulfooxy compounds were deter-
mined by comparison of their 13C‑NMR shifts with those of the
corresponding hydrolysis products. As a consequence of sulfooxy
substitution (K+ salt) the ipso carbon is shifted to high field,
whereas ortho and para carbons are shifted downfield (Table 1S,
Supporting Information).
Table 1 Substituents R5 to R8 of benzopyranones 1 to 15.
Compound
R5
R6
R7
R8
1
2
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OH
H
OCH3
OSO3H
OH
H
3
H
H
4
H
OSO3H
OH
H
Acknowledgements
!
5
OCH3
OH
6
OCH3
H
OH
OSO3H
OH
The authors wish to thank Dr. Karl Schötz for measurement of MS
spectra and Daniela Hauser and Miriam Supper for technical as-
sistance (all Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Ger-
many).
7
OH
8
H
OSO3H
OH
OH
OH
9
H
OH
OSO3H
OSO3H
OH
10
11
12
13
14
15
H
OSO3H
OCH3
OCH3
OH
OH
H
OH
H
OH
OSO3H
OH
References
1 Brendler T, van Wyk BE. A historical, scientific and commercial perspec-
tive on the medicinal use of Pelargonium sidoides (Geraniaceae). J Eth-
nopharmacol 2008; 119: 420–433
H
OCH3
OCH3
OCH3
H
OSO3H
OSO3H
OH
H
OSO3H
2 Bladt S. Zur Chemie der Inhaltsstoffe der Pelargonium reniforme Curt.-
Wurzel (Umckaloabo) [dissertation]. München: Univ. München; 1974
3 Bladt S. Umckaloabo – Droge der afrikanischen Volksmedizin. Dtsch
Apoth Ztg 1977; 117: 1655–1660
4 Produced by ISO-Arzneimittel, Bunsenstraße 6–10, 76275 Ettlingen,
Germany
by spiking the individual authentic reference substances of the
benzopyranones in appropriate amounts to the test solution of
the extract and evaluation of peak characteristics of the resulting
peak.
5 Latté KP, Kayser O, Tan N, Kaloga M, Kolodziej H. Unusual coumarin pat-
terns of Pelargonium species forming the origin of the traditional herb-
al medicine umckaloabo. Z Naturforsch 2000; 55c: 528–533
6 Schoetz K, Erdelmeier C, Germer S, Hauer H. A detailed view on the con-
stituents of EPs® 7630. Planta Med 2008; 74: 667–674
7 Loew D, Hauer H, Koch E. Cumarine in pflanzlichen Arzneimitteln – dif-
ferenzierte Risikobetrachtung. Pharm Ztg 2009; 154: 552–561
8 Hauer H, Ritter T, Grotemeier G. An improved and large scale synthesis
of the natural coumarin scopoletin. Arch Pharm (Weinheim) 1995;
328: 737–738
9 Kayser O. Phenolische Inhaltsstoffe von Pelargonium sidoides DC. und
Untersuchungen zur Wirksamkeit der Umcka-Droge (P. sidoides DC.
und P. reniforme CURT.) [dissertation]. Berlin: Univ. Berlin; 1997
10 Kayser O, Kolodziej H. Highly oxygenated coumarins from Pelargonium
sidoides. Phytochemistry 1995; 39: 1181–1185
11 Hauer H, Jaggy HEW. 7,8-Dihydroxy-5,6-dimethoxy-2H-1-benzopyr-
an-2-one, a new coumarin derived from a labile constituent of Pelargo-
nium sidoides. 49th Annual Meeting of the Society for Medicinal Plant
Research, Erlangen; 2001
12 Goedecke T, Kaloga M, Kolodziej H. A phenol glucoside, uncommon cou-
marins and flavonoids from Pelargonium sidoides DC. Z Naturforsch
2005; 60b: 677–682
Compounds 1 [2,3,5,6,10], 2 [5,6], 3 [5,10], 5 [6,11], 6 [6], 7 [5,6,
10], 8 [6], 9 [6], 10 [6], 11 [12] and 13 [5,6,10] were previously
isolated from Pelargonium sidoides, whereas the known com-
pounds 4 [13] and 15 [6,14] had not been found up to now as
constituents of Pelargonium sidoides or of any other plant. The re-
maining compounds 12 and 14 were identified for the first time.
The HPLC analysis showed that the benzopyranones in Pelargo-
nium sidoides appear mainly as sulfooxy derivatives based on
the six different basic compounds 1 (umckalin), 3 (scopoletin),
5, 7, 11 (fraxetin) and 13. All compounds besides 3 were isolated
from plant material or prepared by hydrolysis of sulfate esters.
Compounds 4 and 15 were additionally prepared by chemical
synthesis from the corresponding hydroxy compounds. Since
the occurrence of 3 in Pelargonium sidoides was known [5,10],
the presence of the corresponding sulfate ester 4 could be ex-
pected. Also it was likely to find the bis(sulfooxy) compound 15
due to the occurence of the compounds 13 and 14, based on the
same phenolic compound.
13 Furuya T. Studies on the metabolism of naturally occurring coumarins.
IV. Urinary metabolites of dimethylesculetin and scopoletin. Chem
Pharm Bull 1958; 6: 696–700
14 Germer S, Hauer H, Koch E, Schoetz K. Use of trisustituted benzopyra-
nones. EP Patent 1763520; 2005
A comparison of the 1H- and 13C‑NMR spectra with published da-
ta showed the identity of the compounds 1 to 3 and 5 to 10. The
1H- and 13C‑NMR spectra of 11 were compared with the corre-
Hauer H et al. Benzopyranones and Their… Planta Med 2010; 76: 350–352

Copyright of Planta Medica is the property of Georg Thieme Verlag Stuttgart and its content
may not be copied or emailed to multiple sites or posted to a listserv without the copyright
holder's express written permission. However, users may print, download, or email articles for
individual use.