Separation of diastereomeric anthrone-C-glucosyls of aloes by micellar electrokinetic capillary chromatography.
ABSTRACT The anthrone-C-glucosyls aloin A and B, 5-hydroxyaloin A, 10-hydroxyaloin A and B were separated by micellar electrokinetic capillary chromatography (MECC) with sodium dodecyl sulfate in borate buffer pH 9 within less than 7 min. The method has been successfully transferred to the analysis of the two European Pharmacopoeia drugs Cape aloes and Curaçao aloes. A comparison of the peak areas received by HPLC and MECC indicated the transferability of the measured contents.
Lehrstuhl Pharmazeutische Biologie, Fakulta ¨t fu ¨r Biowissenschaften,
Pharmazie und Psychologie, Universita ¨t Leipzig, Leipzig, Germany
Separation of diastereomeric
anthrone-C-glucosyls of aloes by micellar
electrokinetic capillary chromatography
T. Liebold, S. Strahler, H. W. Rauwald
Received June 28, 2007, accepted July 20, 2007
Prof. Dr. Hans Wilhelm Rauwald, Institut fu ¨r Pharmazie,
Lehrstuhl Pharmazeutische Biologie, Universita ¨t Leipzig,
Johannisallee 21–23, 04103 Leipzig, Germany
Pharmazie 63: 955–957 (2007)
The anthrone-C-glucosyls aloin A and B, 5-hydroxy-
aloin A, 10-hydroxyaloin A and B were separated by
micellar electrokinetic capillary chromatography (MECC)
with sodium dodecyl sulfate in borate buffer pH 9 within
less than 7 min. The method has been successfully
transferred to the analysis of the two European Phar-
macopoeia drugs Cape aloes and Curac ¸ao aloes. A
comparison of the peak areas received by HPLC and
MECC indicated the transferability of the measured
The term “Aloe” stands for a taxonomic genus of the As-
phodelaceae (Smith et al. 1991). The European Pharmaco-
poeia (Ph.Eur.) lists two drugs, Cape aloes (the dried sap
of the leaves from in particular the species Aloe ferox and
its hybrids) and Curac ¸ao aloes (the dried sap of the leaves
from the species Aloe barbadensis), containing at least
18.0 resp. 28.0 per cent hydroxyanthracene derivatives cal-
culated as aloin. The content of aloin is still specified by
measuring the absorbance after several complicated steps
of extraction, although various simpler methods by HPLC
and capillary electrophoresis (CE) have been developed
during the last years (Suzuki et al. 1986; Rauwald and
Beil 1993a; Kuzuya et al. 2001). The main components of
the two pharmaceutical drugs are the diastereomers aloin
A (10S, 10S) and B (10R, 10S) (Rauwald and Lohse 1989).
Additional native characteristic derivatives (e.g. 5-hydro-
xyaloin A – – naturally occurring in only one configuration
(Rauwald and Beil 1993a) – – in Cape aloes, 7-hydroxy-
aloin A and B in Curac ¸ao aloes (Rauwald and Voetig 1982)
and aloe-emodin in both) are found in minor concentra-
In the analysis of drugs and plants the CE is of increasing
interest (Wa ¨tzig and Dette 1994; Nishi 1999) because of
its many advantages compared to HPLC on certain issues.
Small amounts of buffer and sample, a high separation ef-
ficiency and a short time of analysis are only a few of them.
One method of high potential and easy handling is the mi-
cellar electrokinetic capillary chromatography (MECC).
For some hydroxyanthracene derivatives CE-methods have
been established by several groups (Sheu and Chen 1995;
Sheu and Lu 1995; Weng and Sheu 2000). There are basic
approaches for the separation of the anthrone-C-glucosyls
aloin A and B (Kuzuya et al. 2001), but to our knowledge
neither 5-hydroxyaloin A nor 10-hydroxyaloin A and B,
the main in vitro oxidation products of the aloins (Rau-
wald and Lohse 1992), have been determined by CE yet.
In the present work, we searched for a rapid and easy se-
paration method for the analysis of aloe drugs.
aloin A: R = R’ = H
aloin B: R = R’ = H
R = H
R’ = OH
10-OH-aloin A: R = OH10-OH-aloin B: R = OH
R’ = HR’ = H
With this new method, an optimal separation was obtained
by using a 30 mM borate buffer with 20 mM sodium do-
decyl sulfate (SDS), pH 9. Variant SDS concentrations led
to blurred peaks and worse separation results, in particular,
lower concentrations resulted in current leakage and base-
line drifting. Without the addition of SDS, a separation of
aloin A and B could not be obtained. An increase of pH
up to 10.5 declined the separation. A temperature of 15?C
was associated with baseline and current stability. The sam-
ples were analyzed as shown in the Fig. by typical electro-
pherograms, and their migration times were compared.
The electroosmotic flow (EOF) was detected by using
methanol as marker. 4-Hydroxybenzaldehyde was used as
an internal standard, absorbing at 360 nm. The relative
migration times of aloin A (1.355 ? 0.017) and B
(1.325 ? 0.017) in comparison with 4-hydroxybenzalde-
hyde (1.000) were assayed (n ¼ 9). The 10-hydroxyaloin
sample (Fig. C) contained high quantities of apolar sub-
stances like aloe-emodin and chrysophanol, apart from the
10-hydroxyaloin A and B plus aloin A and B sample, as
it had not been purified after oxidation. The samples of
Cape aloes and Curac ¸ao aloes were compared with the
5-hydroxyaloin A sample. 5-Hydroxyaloin A was found in
Cape aloes, but not in Curac ¸ao aloes. Its migration time
lies between the 10-hydroxyaloins A and B, which have
not been found in those samples. The ratio of aloin B to
A was measured by HPLC (0.749) and compared to that
measured by MECC (0.752).
Aloin A and B were separated by MECC with almost
baseline separation. The generated method allows a simple
determination of anthrone-C-glucosyls in samples, which
is with less than 7 min a lot faster than the established
HPLC methods and to our knowledge faster than the other
MECC methods suggested so far. The higher the polarity
of the substance the lesser it is retarded by interaction
with the negatively charged micelles. Therefore the hydro-
xyaloins show shorter migration times than the aloins. As
diastereomers aloin A and B differ only at C-10 in their
stereochemistry, nevertheless aloin B has a little higher
polarity than aloin A, making the separation with MECC
possible. 4-Hydroxybenzaldehyde has been found to be a
convenient internal standard for anthrone-C-glucosyl se-
Pharmazie 62 (2007) 12955
paration by MECC, as it absorbs with a characteristic
spectrum and is detectable at 360 nm. Its migration time
lies between the examined substances without overlapping
and has reproducible migration time ratios. Because there
is no 5-hydroxyaloin in Curac ¸ao aloes it is possible to dif-
ferentiate the Ph.Eur. drugs by CE. Additional benefit is
the possibility of controlling the progress of 10-hydroxy-
aloin synthesis from the aloins A and B. The comparison
of areas detected by HPLC and CE showed the option to
determine the content of aloe drugs by using this method.
The following HPLC grade chemicals were used: sodium hydroxide solu-
tion 0.1 M (Gru ¨ssing GmbH, Filsum, Germany); sodium hydroxide (La-
chema, Brno, Czech Republic); boric acid, 4-hydroxybenzaldehyde > 98%
for synthesis (Merck, Darmstadt, Germany); methanol LC-MS-grade, so-
dium dodecyl sulfate (SDS) ultra pure ? 99% (Roth, Karlsruhe, Germany).
Water was distilled after deionisation. All samples, except for ‘Aloinum
recrystallized’ Ph.Brit. 1914 (E. Merck, Darmstadt, Germany), were avail-
able in our laboratory: 5-hydroxyaloin A, Cape aloes from Mosselbay and
Curac ¸ao aloes Ph.Eur.III quality; 10-hydroxyaloin was synthesized from
aloin according to Rauwald and Lohse (1992).
CE separations were carried out using a P/ACE? System MDQ (Beckman
Instruments Inc., Fullerton, USA) with a multichannel UV-Vis diode array
detector (Beckman Instruments Inc., Fullerton, USA), software Beckman
P/ACE? System MDQ version 2.3, uncoated fused-silica capillary 75 mm
i.d., 375 mm o.d. eCap? (Beckman Instruments Inc., Fullerton, USA) with
an effective length of 30 cm to the detection window (40 cm total length),
100?800 mm aperture size. The HPLC analyses were performed on an
EC 250/4 Nucleosil? 100-5 c18 column (Macherey-Nagel, Du ¨ren, Ger-
many) with two Dynamax sd-300 pumps (Rainin Instruments, Woburn,
USA). A Dynamax UV1 detector (Rainin Instruments, Woburn, USA) was
used, software: Rainin Dynamax (Rainin Instruments, Woburn, USA).
3. Buffer preparation
For MECC, a 30 mM borate buffer pH 9.0 (made by solving boric acid in
water and titrating it with sodium hydroxide solution 0.1 mM) with an ad-
ditive of 20 mM SDS as surfactant was filtered through a 0.45 mm cellu-
lose nitrate filter (Sartorius, Go ¨ttingen, Germany).
4. Sample preparation
For the analysis of aloin A and B 1.0 ml of methanol was added to 0.5 mg
aloin and then diluted 3 þ 17 with water. 5-Hydroxyaloin (0.45 mg) was
solved in 1.0 ml methanol and then diluted 1 þ 3. The 10-hydroxyaloin
sample was prepared by diluting a 10-hydroxyaloin A and B containing
solution (12.5 mg/ml) 1 þ 19. Cape aloes and Curac ¸ao aloes were ex-
tracted with methanol-water (1 þ 1) each 50.0 mg in 16.7 ml for 15 min,
then filtered through a 0.22 mm membrane filter and diluted 1 þ 5 resp.
3 þ 17. A second time, every sample was prepared with 4-hydroxybenzal-
dehyde (0.05 mg/ml) as internal standard. The samples were proved by
TLC (Rauwald and Beil 1993b); the ratio of aloin A and B in the aloin
sample was measured by HPLC (Rauwald and Beil 1993a).
5. Capillary electrophoresis
Before initiating a series of measurements the capillary was activated by
rinsing 0.1 M NaOH, then water and buffer. Every run was started with
buffer rinsing (2 min, 137.9 kPa); sample injection was performed with
3.4 kPa over 3 s, then separation lasted 7 min (15 kV, 0.17 s ramp time,
average current 25 mA) at 15?C. The substances were detected at 360 nm
and spectra were recorded per PDA-detector to identify the peaks indepen-
dently from their migration time.
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of aloenin, barbaloin and isobarbaloin in aloe species by micellar elec-
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Nishi H (1999) Capillary electrophoresis of drugs: Current status in the
analysis of pharmaceuticals. Electrophoresis 20: 3237–3258.
Rauwald HW, Voetig R (1982) 7-Hydroxyaloin: the characteristic substance
of Aloe barbadensis in the European Pharmacopeia III. Arch Pharm
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der beiden diastereomeren C-Glucosylanthrone Aloin A und B. Ange-
wandte Chemie 101: 1539–1540.
Rauwald HW, Lohse K (1992) Strukturrevision des 4-Hydroxyaloin: 10-
Hydroxyaloine A und B als Haupt-In vitro-Oxidationsprodukte der dia-
stereomeren Aloine. Planta Med 58: 259–262.
956Pharmazie 62 (2007) 12
absorbance (x 10–3)
0.00 2.004.00 6.00
absorbance (x 10–3)
absorbance (x 10–2)
absorbance (x 10–3)
absorbance (x 10–2)
1 2 34
2 31 5 6
Fig.: MECC separation of (A) aloin, (B) 5-hydroxyaloin, (C) 10-hydroxy-
aloin, (D) Cape aloes, (E) Curac ¸ao aloes
(Peak 1: 4-hydroxybenzaldehyde, 2,3: aloin B and A, 4: 5-hydroxy-
aloin A, 5,6: 10-hydroxyaloin B and A);
MECC conditions: capillary: uncoated fused silica capillary (75 mm
?40 cm), temperature: 15?C, UV detection set at 360 nm, applied
voltage: 15 kV, buffer: SDS 20 mM in borate 30 mM, pH 9.0
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Acad. S. Yu. Yunusov Institute of Chemistry of Plant Substances1,
AS RUz, Tashkent, Uzbekistan; Institute of Pharmacy2, Department
of Pharmaceutical Biology, Ernst Moritz Arndt University Greifswald,
New pomolic acid triterpene glycosides from
S. A. Sasmakov1, Zh. M. Putieva1, U. Lindequist2
Received July 5, 2007, accepted August 3, 2007
Prof. Dr. Ulrike Lindequist, Institute of Pharmacy, Ernst
Moritz Arndt University Greifswald, D-17487 Greifs-
Pharmazie 62: 957–959 (2007)
Two new bisdesmosidic triterpenoid saponins, 3-O-[a-
d-glucopyranosyl] ester (zygoeichwaloside H) and 3-O-
[b-d-glucopyranosyl] ester (zygoeichwaloside K) were
isolated from the roots of Zygophyllum eichwaldii. The
structures were established primarily on the basis of
NMR spectroscopy and chemical transformations.
As a part of our continuing phytochemical research on
plants of the genus Zygophyllum which are used in the
traditional medicine of Asian countries (Sasmakov et al.
2001; 2003), this paper deals with the isolation and struc-
tural elucidation of two new triterpenoid saponins from
Separation of triterpene-containing fractions of the methan-
olic extract of the roots of Zygophyllum eichwaldii on a
silica gel column (gradient chloroform, methanol and
water) led to the isolation of two triterpenes (1 and 2). Sa-
ponin 1 was obtained as an amorphous white powder. IR
absorptions at 3410, 1734 and 1648 cm?1indicated the
presence of hydroxyl (OH), ester carbonyl (C¼O), and
double bond (C¼C) functionalities. The olefinic reso-
nances of the aglycone at d 128.05 and 138.97, corre-
sponding to quaternary and methine carbons suggested the
urs-12-ene skeleton with a hydroxyl group at C-19 (Inada
et al. 1987; Ouyang et al. 1997). The13C NMR spectral
data of 1 were consistent with pomolic acid as the agly-
cone (Sasmakov et al. 2001). 41 different signals in the13C
NMR spectrum supported that 1 has a bisdesmosidic struc-
ture (Table). This is confirmed by availability of hydrogen
anomeric atoms at d 5.10 and d 6.32 in the1H NMR spec-
tra. Acidic hydrolysis of the glycoside yielded pomolic
acid as aglycone and arabinopyranose and glucopyranose
as sugar parts. The13C NMR spectrum of 1 contained sig-
nals at d 89.41 attributable to C-3 and showing that the
hydroxyl group at this carbon is glycosylated. The signals
of C-10carbon atom arabinose at d 103.60 and H-10proton
at d 5.10 showed that the arabinose is located at C-3 of the
aglycone. The downfield shifts of the H-20and C-20signals
of arabinose compared with those of Ziyu-glycoside I (3)
(Table) (Sasmakov et al. 2001; Yosioka et al. 1970) indi-
cated that the sulphate group was in position C-20of the
arabinose. The presence of the –SO3H group was con-
Pharmazie 62 (2007) 12957