A detailed view on the constituents of EPs 7630

Article abstract of DOI:10.1055/s-2008-1074515

Extracts of Pelargonium sidoides, commonly known as EPs 7630, are produced by extraction of milled roots with 11 % (w/w) ethanol in water. This solvent leads to a spectrum of constituents which differs significantly from extracts obtained by extraction wi

Full text of DOI:10.1055/s-2008-1074515

Original Paper 667
A Detailed View on the Constituents of EPs® 7630
Author
Karl Schoetz2, Clemens Erdelmeier2, Stefan Germer1, Hermann Hauer2
1
Affiliation
Analytical Development, Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany
Preclinical Research, Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany
2
Key words
Abstract
!
tivities which results in an uncommon diversity
even at a low degree of polymerization. Three
"
● Pelargonium sidoides
"
● Geraniaceae
Extracts of Pelargonium sidoides, commonly known
as EPs® 7630, are produced by extraction of mill-
ed roots with 11% (w/w) ethanol in water. This
solvent leads to a spectrum of constituents which
differs significantly from extracts obtained by ex-
traction with non-polar solvents. EPs® 7630 is
composed of six main groups of constituents,
namely unsubstituted and substituted oligomer-
ic prodelphinidins, monomeric and oligomeric
carbohydrates, minerals, peptides, purine deriva-
tives and highly substituted benzopyranones.
The oligoprodelphinidins, frequently supposed
to be compounds with unspecific tanning inter-
actions, show, in contrast to other polyphenols,
an amazing variety of substructures and connec-
distinct purine derivatives, second messengers
and probably intermediates of DNA synthesis,
were identified and characterized by phytochem-
ical means. The main benzopyranones of EPs®
7630 are highly oxygenated at the phenyl moiety
(three to four oxygens) and in addition sulfated at
distinct positions. A disulfate of 6,7,8-trihydoxy-
benzopyranone has been identified for the first
time in plants. Taken together, these constituents
amount to about 70 to 80% of the total weight of
EPs® 7630, the active ingredient of the phyto-
pharmaceutical Umckaloabo® (Iso Arzneimittel
Ettlingen).
"
● oligomeric prodelphinidins
"
● benzopyranones
"
● sulfates
"
● secondary metabolites
Introduction
which describe constituents of Pelargonium spe-
cies, such as phenols, phenolic acids, benzopyra-
nones, flavonoids and flavan-3-ols as possible ac-
tive ingredients. Unfortunately, these observations
are not always applicable to the therapeutically
used extract EPs® 7630. Reasons for this confusion
include a sometimes unclear assignment of the
plant material, failure to declare if a single plant
genus or a mixture of Pelargonium species has
been investigated, utilization of aerial parts [13]
instead of roots or inappropriate use of non-polar
extraction solvents [14] in place of aqueous etha-
nol. Therefore, it is the aim of this paper to present
a comprehensive profile of the ingredients of EPs®
7630 to indicate which compounds are indeed
contained in the extract and may consequently be
responsible for its activity.
!
Roots of Pelargonium sidoides DC. and P. reniforme
Curt. (family Geraniaceae) are traditionally used in
South African folk medicine for the treatment of a
variety of diseases [1], [2]. At the end of the 19th
century, this ethnomedicine came to Europe more
or less by serendipity and has become a part of the
pool of herbal medicinal products for a long period
of time especially in Germany. Pelargonium ssp.-
containing products have been available for al-
most 100 years. But only the modern phytophar-
maceutical preparation EPs® 7630, a liquid herbal
drug preparation from the roots of Pelargonium
sidoides (1:8–10), extraction solvent: ethanol
11% (w/w), became a successful pharmaceutical
product. It has been used in the treatment of colds
and infections of the respiratory tract during the
last 5–10 years enabled by new phytochemical
[3], [4], pharmacological [5], [6], [7], [8], and clini-
cal studies [9], [10], [11], as well as thorough doc-
umentation of pharmaceutical quality [12]. Over
the last years, several papers have been published
received December 21, 2007
revised March 4, 2008
accepted March 7, 2008
Bibliography
DOI 10.1055/s-2008-1074515
Planta Med 2008; 74: 667–674
© Georg Thieme Verlag KG
Stuttgart · New York
Published online April 30, 2008
ISSN 0032-0943
Correspondence
Dr. Karl Schötz
Preclinical Research
Dr. Willmar Schwabe GmbH &
Co. KG
PO Box D-410925
76209 Karlsruhe
Germany
Tel.: +49/721/4005/536
Fax: +49/0721/4005/8536
karl.schoetz@schwabe.de
Schoetz Ket al. A Detailed View… Planta Med 2008; 74: 667–674

668 Original Paper
Material and Methods
Quantification of peptides
!
Analysis of amino acids (AAs) was performed by HPLC and fluori-
metric detection using a DIONEX P680 pump, DIONEX ASI-100
auto sampler, and a Merck-Hitachi F-1050 fluorescence detector.
Excitation wavelength: 335 nm; emission: 445 nm; column:
Luna (1812) injection volume 100 μL; eluent A: 0.05 M NaH2PO4
pH 5.5/MeOH (80/20; v/v), eluent B: 0.05 M NaH2PO4 pH 5.5/
MeOH (20/80; v/v), gradient: 0 to 63 min, 0 → 100% B; flow rate
0.5 mL/min. Samples dissolved in ethanol/water solutions were
hydrolyzed with 1.5 N HCl at 100 °C for 1.5 h and the reaction mix-
ture neutralized with 10 M NaOH. With a 0.4 M boric acid buffer,
adjusted with NaOH solution to pH 10.4, samples were further di-
luted. AAs were detected after precolumn derivatization with o-
phthaldialdehyde reagent (OPA, 600 μL sample + 240 μL reagent
+ 60 μL 0.4 M boric acid buffer pH 10.4). Quantification was estab-
lished by linear regression with a calibration curve of arginin.
Chemicals and biochemicals
All solvents and chemicals were purchased from VWR or Sigma-
Aldrich with required purity.
Plant material
Roots of Pelargonium sidoides were collected in South Africa
(e.g., Eastern Cape). The dried material was tested in a battery
of phytochemical and biochemical methods to confirm the qual-
ity and identity of the herbal material. Pharmacognosy was done
by P. Riedl [DHU Arzneimittel GmbH & Co. KG, Karlsruhe (Ger-
many)]. A voucher specimen of every lot is deposited in the De-
partment of Pharmacognosy to be kept for ten years.
Extraction and isolation
EPs® 7630 was produced according to Erdelmeier et al. [15].
Quantification of oligoprodelphinidins
Oligoprodelphinidins were quantified by reaction with molyb-
dato reagent according to the described DAB 2000 method for
total phenolics.
LC-DAD-MS analysis
LC-DAD-MS data were obtained with an Agilent 1100 series
HPLC system consisting of an auto sampler, high pressure mixing
pump, column oven and DAD detector connected to a Bruker Es-
quire HCT ion trap mass spectrometer. HPLC conditions: col-
umn: Phenomenex LUNA and Synergy, respectively; solvent sys-
tem: A–3% MeOH in water + 1% acetic acid, B–70% MeOH + 1%
acetic acid; gradient: 0–5% B for 5 min, 5–100% B for 40 min,
100% B for 15 min; flow rate 1 mL/min; injection volume: up to
50 μL, sample concentration: about 1 mg/mL in eluent A. ESI
conditions: alternating polarity with MS2 fragmentation of the
two most intensive signals.
Purine derivatives
500 g dried roots from Pelargonium sidoides were extracted with
5 kg 11.2% aqueous ethanol. The mixture was filtered, the filtrate
evaporated under vacuum to remove the organic solvent. The re-
maining water phase was freeze-dried. An aliquot (50 g) of the
lyophilisate was dissolved in water, filtered over HP20, and the
filtrate chromatographed on PVP. Fractions containing the pu-
rines were concentrated and the anions adsorbed on a strongly
basic ion exchanger. The resin was eluted with 1 N hydrochloric
acid, the eluate neutralized (pH 5) and freeze-dried. The residue
was taken up in MeOH, the suspension filtered, and the filtrate
evaporated to remove the organic solvent. The remaining aque-
ous phase was separated by HPLC (column: Hibar Lichrospher
100, RP-18; eluent: water/acetonitrile/acetic acid (99:1:1; v/v/
v); detection: UV 254 nm).
NMR spectroscopy
1H- and 13C-NMR spectra were acquired on a Bruker DPX200
spectrometer (200 MHz) in DMSO-d6 unless otherwise men-
tioned. The chemical shifts refer to TMS (1H NMR) or to DMSO-
d6 (13C NMR, δ = 39.5 ppm), respectively.
Quantification of minerals
cAMP: 185 mg (0.037% isolated from dried roots, 0.64% of EPs®
7630 by HPLC analysis); was identified by matching 1H- and
13C-NMR data of Aldrich-Library 3,229 A and commercially
available material (Sigma A-9501 lot 22K1762).
cGMP: 240 mg (0.048% of dried roots, 0.68% of EPs® 7630), was
identified by comparison of 1H- and 13C-NMR data with those of
commercially available material (Fluka 51055 lot 402854/1
34502041).
1-Methyl-cGMP: 85 mg (0.017% of dried roots, 0.35% of EPs®
7630); was identified by analysis of 1H- and 13C-NMR data (data
are not available in the literature). The extra methyl group was
located by an HMBC experiment. 1H NMR (NaOD/D2O, 200 MHz):
δ = 3.44 (3H, s, 1-Me, HMBC to C-2, C-6), 4.22–4.68 (6H, m, H-2′
to H-5′), 5.82 (1H, s, H-1′, HMBC to C-4, C-8), 7.87 (1H, s, H-8,
HMBC to C-4, C-5, C-6); 13C NMR (NaOD/D2O, 50 MHz): δ = 31.4
(CH3, 1-Me), 70.5, 70.6 (CH2, C-5′), 74.9, 75.0 (CH, ribosyl-H),
77.5, 77.6 (CH, ribosyl-H), 82.0, 82.1 (CH, ribosyl-H), 97.4 (CH, C-
1′), 118.8 (C, C-5), 140.8 (CH, C-8), 151.8 (C, C-4), 157.6 (C, C-2),
161.5 (C, C-6).
Sodium, magnesium, potassium, calcium and several other met-
als were analyzed by ICP-MS-AES at Mikroanalytisches Labor
Pascher, An der Pulvermühle 1, D-53424 Remagen–Bandorf,
Germany.
Quantification of carbohydrates by HPLC-RI
Hydrolysis: A suspension of 2 mg extract in 400 μL 2 M trifluoro-
acetic acid (TFA) in water was heated in sealed glass vials to
120 °C for up to 90 min. The reaction mixture was three times
evaporated by a nitrogen stream under sequential addition of
water to remove all TFA. Finally, the residue was taken up in
800 μL of water, filtered, and the carbohydrates quantified by
HPLC. Column: Rezex RPM Monosaccharide, 300×7.8 mm,
8 μm, ion form: Pb2+, order Nr. Phenomenex OOH-0135-KO and
precolumn AJO-4492, 4×3 mm; eluent: distilled water; flow
rate: 0.6 mL/min; column temperature: 75 °C; detection: refrac-
tive index detector.
Quantification of monosaccharides by anthrone colour
reaction
The method is described in Ref. [16].
Fractionation of EPs® 7630
Ultrafiltration: 400 g EPs® 7630 were taken up in 710 g water and
996 g 96% EtOH, the suspension filtered (Seitz Supra 1500), and
the solution submitted to successive steps of ultrafiltration
across membranes with molecular cut-offs of 30 kDA, 10 kDa
Schoetz Ket al. A Detailed View… Planta Med 2008; 74: 667–674

Original Paper 669
and 3 kDa. The final filtrate was evaporated on a vacuum evapo-
rator to remove the organic solvent and finally freeze-dried
(130 g, 32.5%).
Mixed ammonia/potassium salt (5c): 28.25 g of the above ultra-
filtrate were chromatographed on 480 g LH20 (5 cm×92 cm) us-
ing 1% MeOH in water as eluent. Fractions of approx. 50 mL
were collected, analyzed by TLC (ethyl acetate/acetic acid/formic
acid/water = 50:11:11:27). Fractions of similar composition
were combined. The fraction containing the compound at
Rf = 0.6 (vials 39 to 43) was freeze dried and the residue extrac-
ted 2 times with 20 mL 50% MeOH to remove polar impurities.
The formed solid was collected on a glass frit, washed two times
with 20 mL MeOH and finally dried at 40°C in vacuo to give
Benzopyranones
Milled dried roots from Pelargonium sidoides (15 kg) were ex-
tracted with water. The aqueous extract was saturated with am-
monium sulfate and extracted three times with 2-butanone/
ethanol (3:2; v/v). The combined organic phases were evapora-
ted, the residue taken up in water and the solution chromato-
graphed over HP20 by reducing the polarity of the eluent to
10% ethanol. For further separation of the compounds 1, 2, 3, 4,
5a, 12a and 12b, the fractions were treated as described below.
7-Hydroxy-5,6-dimethoxy-2H-1-benzopyran-2-one (umckalin, 1):
The fractions containing 2 were concentrated and decomposed
to 1 at elevated temperature (50 °C, 3 h), washed with water
and recrystallized from water/methanol: yield: 18.1 g, m.p.147
– 148.5°C (146 – 147 °C, Ref. [11]); 1H- and 13C-NMR in acetone-
d6 corresponded to the data of Ref. [17]. For convenience NMR
1
13
"
250 mg (0.9%) of disulfate 5b. H- and C-NMR see ● Table 3;
ESI-MS (pos. ion mode): m/z = 355.0 → 275.0 → 194.9; ESI-MS
(neg. ion mode): m/z = 272.9 → 192.9; elemental analysis, calcd.
as mixed ammonia potassium salt: C 25.53, H 1.27, K 15.39, N
1.10, S 15.1; found: C 25.28, H 1.56, K 15.7, N 0.84, S 13.3.
6,7-Dihydroxy-8-(sulfooxy)-2H-1-benzopyran-2-one (12a) and
7,8-dihydroxy-6-(sulfooxy)-2H-1-benzopyran-2-one (12b): The
fractions containing 12a and 12b were adjusted to pH 7.5, con-
centrated and purified by column chromatography on LH-20
(methanol). The mono potassium salts of 12a and 12b were sep-
arately crystallized from methanol to afford 435 mg of 12a and
"
values are presented in DMSO-d6 in ● Table 2.
5,6-Dimethoxy-7-(sulfooxy)-2H-1-benzopyran-2-one (umcka-
lin sulfate, 2): The fractions containing 2 and 4 were adjusted
to pH 7.5 with diluted potassium hydroxide solution and con-
centrated. Purification was performed by column chromatogra-
phy on Sepabeads Sp-825 (water → 20% ethanol), adjustment
to pH 7.5 again and a further chromatography on LH-20 (metha-
nol) gave the monopotassium salt of 2, which was recrystallized
from ethanol/water: yield: 8.5 g slightly yellow powder. 1H- and
285 mg of 12b. 1H- and 13C see ● Table 3.
"
6,7,8-Trihydroxy-2H-1-benzopyran-2-one (13): Crude compound
5a (20 g) was dissolved in hot water, filtered, acidified with HCl
and stirred for 20 h at 40 to 50°C. Compound 13 precipitated on
cooling and was recrystallized from water; yield: 5.9 g; m.p.
260°C (dec.; ref. 20: 258°C); 1H- and 13C-NMR in acetone-d6 cor-
respond to those in Ref. [11].
13
"
C-NMR (DMSO-D6) see ● Table 2 (Ref. [18] gives NMR data in
6,8-Bis(sulfooxy)-7-methoxy-2H-1-benzopyran-2-one (6): 38 mg
5b (0.088 mmol) were dissolved in 4 mL 10% MeOH in water and
successively treated with a 200-fold excess of 1 M diazomethane
in ether. The reaction mixture was washed twice with 1 mL di-
ethyl ether and freeze-dried, the residue agitated with 2 times
10 mL isopropanol, and the solid residue dried under vacuum at
40°C to afford 6; yield: 34 mg (86.7%); elemental analysis, calcd.
as dipotassium salt monohydrate: C 25.97, H 1.74; found: C
CD3OD).
7,8-Dihydroxy-5,6-dimethoxy-2H-1-benzopyran-2-one (3): The
fractions containing 4 were concentrated and decomposed to 3
at elevated temperature (50 °C, 3 h). Purification was performed
by column chromatography on HP-20 (20% ethanol → 35% etha-
nol) and on Sepabeads Sp-850 (10% ethanol → 30% ethanol).
Crude 3 was washed with ethanol and recrystallized from water:
1
yield: 2.4 g, m.p. 202–203 °C. H- and 13C-NMR (DMSO-d6) see
26.09, H 2.01; 1H- and 13C-NMR see ● Table 3.
"
"
● Table 2.
6,8-Dihydroxy-7-methoxy-2H-1-benzopyran-2-one (7): 33 mg
(0.074 mmol) 6 were dissolved in 1% aqueous KHSO4 and heated
for 7 h at 100°C. The reaction mixture was extracted 3 times
with 20 mL ethyl acetate, the combined organic phases washed
with 10 mL saturated NaCl solution, dried over Na2SO4 and
evaporated to afford 7; yield: 14 mg (90.6%); 1H- and 13C-NMR
7-Hydroxy-5,6-dimethoxy-8-(sulfooxy)-2H-1-benzopyran-2-one
(4): Chromatography as described for compound 2 afforded the
monopotassium salt of 4: yield: 4.4 g yellow crystals. 1H- and
13
"
C-NMR see ● Table 2
8-Hydroxy-5,6,7-trimethoxy-2H-1-benzopyran-2-one: To define
the position of the sulfate group in 4, the compound was methy-
lated with MeI/K2CO3 in DMF at 60 °C for 1 h. The intermediate
sulfate was saponified by addition of concentrated HCl, the re-
sulting phenol extracted with ethyl acetate, and the organic sol-
vent evaporated under vacuum. The product was finally purified
by chromatography over silica (eluent: ethyl acetate) and crys-
tallization from methanol. 1H- and 13C-NMR data were identical
to those of Ref. [19].
"
see ● Table 3.
6,8-Dibenzyloxy-7-methoxy-2H-1-benzopyran-2-one (8): 1H-NMR
(CDCl3): δ = 7.55 (d, J = 9.5 Hz, H-4), 7.58 – 7.34 (m, 2×C6H5), 6.72
(s, H-5), 6.32 (d, J = 9.5 Hz, H-3), 5.24 (s, 8-OCH2), 5.13 (s, 6-
OCH2), 3.95 (s, 7-OCH3); 13C-NMR (CDCl3): δ = 160.3 (C-2), 149.1
(C-6), 147.0 (C-7), 143.5 (C-8a), 143.3 (C-4), 140.1 (C-8), 136.8,
136.3 (C-1′, C-1′′), 128.7, 128.5, 128.4, 127.3 (C-2′/6′, C-3′/5′, C-
2′′/6′′, C-3′′/5′′), 128.24, 128.20 (C-4′, C-4′′), 115.2 (C-3), 114.3 (C-
4a), 106.4 (C-5), 76.0 (8-OCH2), 71.5 (6-OCH2), 61.6 (7-OCH3).
Methyl 3′,5′-bis(sulfooxy)-2′,4′-dimethoxycinnamate (10): 530 mg
of disulfate 5b (1.18 mmol) were dissolved in 50 mL 10% MeOH
in water and successively treated with a 200-fold excess of 1 M
diazomethane in ether. The water phase was washed 3 times
with 25 mL diethyl ether and then freeze-ried (620 mg). 250 mg
of crude product were chromatographed over 80 g LH20 (1.6×
41 cm, about 7 mL per fraction) using water as eluent. Fractions
27 and 28 were combined and freeze-dried to afford compound
10; yield: 77 mg (13.3%); elemental analysis, calcd. as dipotassi-
um salt dihydrate: C 27.37, H 3.06; found: C 27.31, H 2.77; 1H-
6,8-Bis(sulfooxy)-7-hydroxy-2H-1-benzopyran-2-one (5a and
b,c)
Tripotassium salt 5a: The fractions containing 5 were adjusted to
pH 10.7 with dilute potassium hydroxide solution, and diluted
with ethanol. The precipitate was collected, washed with etha-
nol/water 1/1 and dried. The tripotassium salt of 5a (yield:
33.2 g) was obtained. 1H- and 13C-NMR see ● Table 3.
"
Dipotassium salt 5b: 5a was dissolved in water, adjusted to pH 7
and diluted with ethanol. The solid was filtered off, washed with
ethanol/water 1/1 and dried to afford the dipotassium salt of 5b.
1H- and 13C see ● Table 3.
"
Schoetz Ket al. A Detailed View… Planta Med 2008; 74: 667–674

670 Original Paper
and 13C-NMR see ● Table 4; ESI-MS (pos. ion mode): m/z = 415.1
→ 383.0 →303.1 → 223.0; ESI-MS (neg. ion mode): m/z = 413.0 →
333.1 → 252.9 → 238.3.
detected with electrospray MS, see ● Fig. 3) and similar connec-
"
"
tivities can be expected to be contained in EPs® 7630 indicating
that Pelargonium sidoides prodelphinidins represent a rich diver-
sity of structures.
E- and Z-Methyl 3′,5′-dihydroxy-2′,4′-dimethoxycinnamate (11):
A solution of 30 mg (0.060 mmol) 10 dissolved in 15 mL 3%
MeOH was acidified by addition of 0.75 mL acetic acid and kept
7 h at 100°C. Afterwards the reaction mixture was extracted
with 50 mL TBME, the organic phase washed 2 times with
The second large group of EPs® 7630 constituents are typical
members of the primary plant metabolism, which are extracted
because of the very polar conditions employed. To enable the
quantification of carbohydrates, amino acids and peptides, and
minerals, extracts were heated in acidic media to yield quantifi-
able low molecular weight compounds. In case of carbohydrates,
monosaccharides like glucose, galactose, mannose, and fructose
as well as inositol and mannitol were individually quantified by
HPLC-RI. In addition, carbohydrates were quantified by photo-
metry after a colour reaction with anthrone. Both assays gave a
total amount of 10 – 12% carbohydrates in the dry extract.
Minerals and inorganic salts contribute to the composition of
EPs® 7630 in a comparable amount. Cations were detected as
30 mL water, dried over Na2SO4 and evaporated to afford 11;
yield: 5.5 mg (36.0%); 1H- and C-NMR of 11a see ● Table 4;
13
"
1H-NMR 11b (DMSO-d6, 600 MHz): δ = 3.68 (3H, s, 2′-OCH3),
3.72 (3H, s, 1-OCH3), 3.74 (3H, s, 4′-O CH3), 6.34 (1H, d,
J = 16.2 Hz, H-2), 6.62 (1H, s, H-6′), 7.72 (1H, d, J = 16.2 Hz, H-3),
9.04, 9,18 (2H, broad, Ar-OH); ESI-MS (pos. ion mode): m/z =
277.1 → 223.0 → 207.9; ESI-MS (neg. ion mode): m/z = 252.9 →
237.9 → 222.9.
Results and Discussion
!
EPs® 7630 is produced by extraction of dried roots of
Pelargonium sidoides with 11% aqueous ethanol. This leads to a
specific mixture of polar compounds derived from the primary
"
and secondary metabolism of the plant (● Fig. 1).
About 40% of the dried extract can be attributed to oligomers of
the proanthocyanidin family, mainly based on gallocatechin and
epigallocatechin entities connected by B- and A-type bonds
"
(● Fig. 2). Additionally, some oligomers show mono-derivatiza-
tion at as yet undefined positions [4] of the phenolic functions.
The detected isomers of oligoprodelphinidin dimers implement
a diversity of extraordinary broadness. It seems that at least all
"
currently known stereoisomers (● Table 1) are present in the ex-
tract. Additional isomers may be present but still await structure
elucidation [4]. Oligomers of higher masses (up to 16 mer can be
Fig. 2 General structures of B- and A-type oligomeric prodelphinidins.
Fig. 1 Compositional profile of EPs® 7630.
Table 1 Constitutions of
known prodelphinidin dimers
(for numbering see Fig. 2)
B-type bonding
A-type bonding
Name
Configuration
Name
Configuration
Prodelphinidin B1
Prodelphinidin B2
Prodelphinidin B3
Prodelphinidin B4
Prodelphinidin B9
2R,2′R,3R,3`S,4R
2R,2′R,3R,3′R,4R
2R,2′R,3S,3′S,4S
2R,2′R,3S,3′R,4S
2R,2′R,3R,3′R,4S
Prodelphinidin A1
Prodelphinidin A2
3′R
3′S
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Original Paper 671
Fig. 3 Infusion electrospray mass spectrum of a
prodelphinidin polymer. The electrospray signals
are composed of singly charged (black colour, n-),
doubly charged (blue colour; n2–), and triply charg-
ed ions (red colour; n3–). Therefore, three different
classes of overlapping signals are visible. Signals at
m/z = 912.5, 1217.1, and 1521.1 are assembled
from singly, doubly and triply charged ions. Signals
at m/z = 760.1, 1064.7, 1368.6, and 1672.5 show
doubly charged species. Signals at m/z = 810.6,
1013.6, 1150.0, 1318.1, 1419.3, and 1622.5 are
assignable to triply charged oligomers. From all this
data results a repeating unit of 304 Da (dehydro-
prodelphinidin), the mass difference between sin-
gly charged ions.
Fig. 4 HPLC-UV chromatogram of purine deriva-
tives. Column: Phenomenex Synergy 4 μm Hydro-
RP 150×2 mm; Eluent: MeCN/H2O/HCOOH =
5:95:0,3; isocratic: 10 min; flow 0.2 mL/min; de-
tection: UV 254 nm.
such with ICP-MS: potassium (∼4%), sodium (∼1.2%), and mag-
nesium (∼0.4%). Approx. 40 transition metals were analyzed
but were below 0.1%. Anions were quantified by ion chromatog-
raphy giving sulfate (∼4.5%), phosphate (∼2%), and chloride
(∼1%). All ions together add up to 10 to 14%. This was in good
agreement with combustion experiments, where a total mineral
content of 10 to 12% was found.
The most characteristic secondary metabolites of Pelargonium
sidoides are benzopyranones, especially umckalin 1 [14]. Other
members of this group include 5,6-dimethoxy-7,8-dihydroxy-
coumarin 3 and 6,7,8-trihydroxycoumarin 13. A remarkable fea-
ture is that predominant amounts of these compounds occur as
"
their sulfated derivatives (● Fig. 5).
Peptides, amino acids and their derivatives constitute another
substantial part of EPs® 7630. Main components detected after
hydrolysis include arginine (Arg: ∼4%), glutamine/glutamic acid
(Glx: ∼2%) and asparagine/aspartic acid (Asx: ∼2%), whereas the
other amino acids together sum up to about 2% of the dried ma-
terial.
Less abundant constituents of EPs® 7630 which, however, can
easily be detected in the UV-chromatogram at 254 nm
"
(● Fig. 4), are purine derivatives. Along with cAMP [21] and
cGMP [22], a typical second messenger more often known from
bacterial and mammalian cells, 1-methyl-cGMP, was identified
as a quite uncommon derivative. This compound has not been
reported from plants before [23]. Overall, these compounds
amount up to almost 2% of dried EPs® 7630.
Fig. 5 Structures of major 5,6,7-trihydroxybenzopyran derivatives and
their sulfates.
Schoetz Ket al. A Detailed View… Planta Med 2008; 74: 667–674

672 Original Paper
Fig. 6 Derivatization reactions for the structure elucidation of 5.
Table 2 1H- and 13C-NMR
shifts (1H with multiplicity and
coupling constants) of the
benzopyranones 1 – 4
Compound
1
2a
6.32 (d, 9.7)
3
4a
6.07 (d, 9.5)
H-3
6.19 (d, 9.7)
6.20 (d, 9.7)
H-4
7.96 (dd, 9.7, 0.5)
3.94 (s)
3.77 (s)
10.64 (s)
6.60 (d, 0.5)
–
8.02 (d, 9.7)
3.94 (s)
3.81 (s)
–
7.97 (d, 9.7)
3.84 (s)
3.78 (s)
9.6 (br. s)
–
7.91 (d, 9.5)
3.88 (s)
3.79 (s)
9.95 (br. s)
–
5-OCH3
6-OCH3
7-OH
H-8
7.30 (s)
–
8-OH
C-2
9.6 (br. s)
160.2
–
160.3
160.1
113.2
138.9
108.3
149.5
61.9
160.4
109.2
139.5
103.5
145.7
61.9
C-3
111.1
111.2
C-4
139.3
139.6
C-4a
C-5
105.6
105.0
149.3
141.5
5-OCH3
C-6
61.8
62.1
137.2
139.6
61.0
137.8
138.7
60.3
6-OCH3
C-7
60.5
60.6
155.5
151.0
103.2
148.9
144.4
152.7
125.6
144.6
C-8
98.7
129.3
C-8a
150.8
139.6
a Sulfates 2 and 4 were measured as potassium salts.
All spectra were recorded in DMSO-d6.
A new derivative that appeared in standard chromatograms at
very low retention times was characterized by mass spectrosco-
py and elemental analysis as a disulfate. Its structure was eluci-
of some ammonia (5c) leads to chemical shift differences (see
● Table 3) that were not expected from less polar compounds.
An extreme change in chemicals shifts is observed in the tripo-
tassium salt 5a that was isolated at pH ∼11. Thus, spectral inter-
pretation for compounds must be taken with care as long the
number and character of counterions is not known.
"
"
dated by chemical and spectroscopic means (● Fig. 6). Reaction
of the compound with diazomethane introduced a methyl group
and located the free phenol function. Hydrolysis of both sulfate
groups afforded 7. The two phenolic groups were successively al-
kylated with benzyl bromide. NOE correlation spectroscopy
showed spatial proximity of the benzylic methylene protons
with the methoxy group at C-6 [15].
Additional benzopyranones 12 and 13 were products of stepwise
"
removal of sulfate moieties (● Fig. 6). Although the sulfated
benzopyranones show very prominent and characteristic signals
in HPLC-UV chromatograms, only about 2% of dry weight is at-
tributed to this class of compounds.
EPs® 7630 is a modern herbal medicinal product with demon-
strated clinical effectiveness [24], [25], [26], [27], [28], [29],
[30], [31]. According to present investigations, the oligomeric
prodelphinidins, benzopyranones, carbohydrates, minerals, ami-
no acids and peptides, and purines add up to 70 to 80% of the dry
matter of the extract, leaving 30 to 20% of yet unknown constit-
uents. Although secondary metabolites are generally believed to
Methylation of 5 under slightly basic conditions afforded a cin-
namate 10, which in turn lead to dihydroxy-dimethoxy deriva-
tive 11 after removal of the sulfate groups. HMBC correlation
spectroscopy confirmed the proposed substitution pattern (see
"
● Table 4). The compound forms a stable mixture of Z- and E-
isomers (11a and 11b) in a ratio of 3:1. It is important to notice
that the 1H- and 13C-NMR data of compound 5 are extremely
sensitive to changes in the ion sphere. Already the incorporation
Schoetz Ket al. A Detailed View… Planta Med 2008; 74: 667–674

Original Paper 673
Table 3 1H- and 13C-NMR shifts (1H with multiplicity and coupling constants) of the benzopyranones 5 – 13
Com-
5aa
5ba
5ca
6
7
12ab
12bb
13
pound
H-3
5.59 (d, 9.1)
6.11 (d, 9.4)
5.90 (d, 9.3)
6.31 (d, 9.5)
6.31 (d, 9.5)
6.23 (d 9.5)
7.89 (d, 9.5)
6.86 (s)
6.22 (d, 9.5)
7.92 (d, 9.5)
7,09 (s)
6.17 (d, 9.5)
7.84 (d, 9.5)
6.56 (s)
H-4
7.62 (d, 9.1)
7.03 (s)
7.91 (d, 9.4)
7.43 (s)
–
7.82 (d, 9.3)
7.26 (s)
–
7.99 (d, 9.5)
7.55 (s)
–
7.88 (d, 9.5)
6.59 (s)
H-5
6-OH
7-OH
7-OCH3
8-OH
C-2
9.54 (s)
9.55 (br s)
9.55 (br s)
9.41 (s)
9.43 (br s)
9.43 (br s)
9.41 (s)
–
–
–
–
3.92 (s)
–
3.78 (s)
9.70 (s)
9.41 (s)
161.9
160.5
161.1
160.1
160.2
160.3
112.4
144.5
111.0
108.7
143.8
143.4
–
160.4
160.6
C-3
102.4
145.1
101.5
115.5
142.2
162.9
–
110.5
144.9
108.7
115.1
140.0
149.6
–
107.0
145.0
105.7
115.3
140.9
n. d.
113.9
144.4
113.1
114.8
143.2
148.9
60.8
114.3
144.6
114.5
103.4
147.3
139.6
60.2
112.1
145.0
110.5
111.0
138.4
142.1
–
111.6
145.0
110.4
103.2
142.9
138.9
C-4
C-4a
C-5
C-6
C-7
7-OCH3
C-8
–
147.7
130.4
128.7
144.8
129.5
146.0
133.6
144.7
138.2
137.1
128.4
141.7
133.5
140.5
132.9
138.1
C-8a
a The disulfates 5a – c were measured as potassium salts containing different phenolic counterions; 5a was a potassium phenolate, 5b the free phenol, and 5c a mixed
phenol/ammonium phenolate form.
b Sulfates 12a and 12b were measured as potassium salts.
All spectra were recorded in DMSO-d6.
Table 4 1H- and 13C-NMR shifts (1H with multiplicity and coupling constants),
and HMBC correlations of cinnamic acid derivatives 10 + 11b
#
10
11a
5.95; d; 12.6
H-2
5.95 d, 10.5
6.97 d, 10.5
7.36, s
3.64 s
3.77 s
3.83 s
–
H-3
6.97; d; 12.6
6.55; s
H-6′
1-OCH3
4′-OCH3
2′-OCH3
5′-OH
3′-OH
C-1
3.63 s
3.72 s
3.60 s
8.81 br
–
9.00 br
166.5 (H-3; 2-OCH3)
118.9 (H-3)
136.7 (H-5)
121.5 (H-3)
148.8 (H-4, H-5, 8a-OCH3)
139.2
166.4 (2-OCH3)
119.0 (H-3)
136.7 (H-5)
112.8 (H-3)
143.3 (8a-OCH3)
137.5 (8-OH, 6-OH)
145.9 (7-OCH3)
139.9 (8-OH)
106.3 (H-5, H-4; 6-OH)
51.1
C-2
C-3
C-1′
C-2′
C-3′
C-4′
C-5′
C-6′
1-OCH3
2′-OCH3
4′-OCH3
147.5(H-5, 7-OCH3)
141.4 (H-5)
118.6 (H-5, H-4)
51.3
Fig. 7 Hydrolysis of disulfate 5.
60.5
60.7
60.8
59.8
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unique combination of bioactive molecules and a matrix of pri-
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