Urinary excretion of arbutin metabolites after oral administration of bearberry leaf extracts

Article abstract of DOI:10.1055/s-2005-837782

An HPLC assay with fluorimetric detection of the arbutin metabolites hydroquinone glucuronide (2) and hydroquinone sulphate (6) in urine was developed and validated. Methylarbutin (4) and 6 were synthesised as reference substances. Compound 2 was prepared enzymatically from hydroquinone and uridine 5-diphosphoglucuronic acid using the glucosyltransferase system of rat liver microsomes and enriched by two liquid-liquid and an additional solid phase extraction. Compound 2 as the main component of this purified product was identified by UV and fluorescence spectroscopy, by HPLC-MS, and by enzymatic hydrolysis to hydroquinone (5). The assay yields precise and accurate urine levels of 2, 5 and 6 in the concentration range expected after oral administration of recommended therapeutic doses of bearberry leaf extract. In a preliminary pharmacokinetic study on 3 volunteers the time-dependent renal excretion of arbutin metabolites 2, 5 and 6 was investigated after ingestion of an aqueous bearberry leaf extract containing an arbutin dose recommended by the German Kommission E. More than half of the administered dose of arbutin was excreted within 4 hours mainly in form of the metabolites 2 and 6 and more than 75% of the total applied arbutin was excreted within 24 h. The elimination of 5 was negligible in 2 out of 3 volunteers. The excretion of this metabolite in the third test person reached 5.6% of the total administered arbutin dose. The preliminary pharmacokinetic results confirm that renal elimination of toxicologically critical concentrations of the metabolite 5 will not be expected.

Full text of DOI:10.1055/s-2005-837782

Joachim Quintus1
Karl-Artur Kovar1
Peter Link2
Urinary Excretion of Arbutin Metabolites after Oral
Administration of Bearberry Leaf Extracts
Harald Hamacher2
Abstract
study on 3 volunteers the time-dependent renal excretion of ar-
butin metabolites 2, 5 and 6 was investigated after ingestion of
An HPLC assay with fluorimetric detection of the arbutin meta- an aqueous bearberry leaf extract containing an arbutin dose re-
bolites hydroquinone glucuronide (2) and hydroquinone sul- commended by the German Kommission E. More than half of the
phate (6) in urine was developed and validated. Methylarbutin administered dose of arbutin was excreted within 4 hours mainly
(4) and 6 were synthesised as reference substances. Compound in form of the metabolites 2 and 6 and more than 75% of the total
2 was prepared enzymatically from hydroquinone and uridine applied arbutin was excreted within 24 h. The elimination of 5
5¢-diphosphoglucuronic acid using the glucosyltransferase sys- was negligible in 2 out of 3 volunteers. The excretion of this me-
tem of rat liver microsomes and enriched by two liquid-liquid tabolite in the third test person reached 5.6% of the total admin-
and an additional solid phase extraction. Compound 2 as the istered arbutin dose. The preliminary pharmacokinetic results
main component of this purified product was identified by UV confirm that renal elimination of toxicologically critical concen-
and fluorescence spectroscopy, by HPLC-MS, and by enzymatic trations of the metabolite 5 will not be expected.
hydrolysis to hydroquinone (5). The assay yields precise and ac-
curate urine levels of 2, 5 and 6 in the concentration range ex- Key words
pected after oral administration of recommended therapeutic Arctostaphylos uva-ursi ´ Ericaceae ´ bearberry leaf ´ arbutin ´
doses of bearberry leaf extract. In a preliminary pharmacokinetic bioanalytics ´ pharmacokinetics
147
Introduction
is specifically determined by a HPLC assay. Compound 1 is a pro-
drug. After oral administration it is demethylated and metabo-
Uncomplicated infections of the urinary tract can be treated lised and finally renally eliminated as hydroquinone conjugates.
using diuretic and anti-infective herbal drug preparations. The
anti-infective herbal drug most used is bearberry leaf from The mechanism of the absorption of 1 is not quite clear. In con-
Arctostaphylos uva-ursi (L.) Spreng. which is recognised by the trast to the assumption communicated in text books [1], [2] that
German registration authority. The monograph bearberry leaf in 1 is absorbed as its aglycone 5 after hydrolysis by b-glucosidases
the European Pharmacopoeia, 5th edition, specifies a minimum of the intestinal microbial flora, experimental investigations sug-
content of 7.0% of arbutin (1; hydroquinone 1-O-b-D-glucopyra- gest an active transport of intact 1 mediated by the glucose carri-
noside), calculated on the dried drug, as the accepted therapeu- er system [3], [4]. Even in earlier papers, e.g., by Frohne [5],
tically relevant component which, in contrast to earlier editions, transformation of 1 and elimination in urine as 2 and 6 was sug-
Affiliation
1 Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Tübingen, Germany
2 LAZ - Laboratorium für Arzneimittelprüfung und Zulassungsberatung, Tübingen, Germany
Correspondence
Prof. Dr. Harald Hamacher ´ LAZ ± Laboratorium für Arzneimittelprüfung und Zulassungsberatung ´
Dischingerweg 15´ 72070 Tübingen ´ Germany ´ Phone: +49-7071-791163 ´ Fax: +49-07071-791598 ´
E-mail: LAZ-Prof.Hamacher@t-online.de
Received April 13, 2004 ´ Accepted August 22, 2004
Bibliography
Planta Med 2005; 71: 147±152 ´ ꢀ Georg Thieme Verlag KG Stuttgart ´ New York
DOI 10.1055/s-2005-837782
ISSN 0032-0943

Equipment
Melting points were obtained according to Ph. Eur. 2.2.14 (capil-
lary method), 1H-NMR spectra on a Bruker AC 250 (Bruker Bio-
Spin, Rheinstetten, Germany), mass spectra were obtained with
fast atom bombardment on a Finnigan TSQ 70 (Thermo Electron,
Dreieich, Germany) and FTIR spectra on a Paragon 500 (Perkin-
Elmer, Wiesbaden, Germany). HPLC used for the purity control
of reference substances and the bioanalytical investigations
were performed on a LaChrom L-7000 series (Merck-Hitachi,
Darmstadt, Germany). HPLC-MS experiments were performed
with an Agilent 1100 Series (Agilent, Böblingen, Germany) in
combination with an esquire 3000plus (Bruker Daltronik, Bre-
men, Germany) using ESI in the positive mode. Stationary phases
for assays of bearberry leaf extracts were: Nucleosil 120 C18 5 mm
Macherey-Nagel, Düren, Germany (precolumn: 8 mm ” 4 mm;
columns: 250 mm”4 mm) assays of arbutin metabolites in
urine: ReproSil Pur C18AQ 5 mm, Dr. Maisch, Herrenberg, Germa-
gested, but not proved. Frohne [5] applied a conventional unspe- ny (precolumn: 5mm”4.6 mm; column: 250 mm”4.6 mm).
cific spectrophotometric assay to determine arbutin metabolites
in urine. This unspecific spectrophotometric method was also Preparation of reference substances 2, 4 and 6
applied for determining elimination products of arbutin in a re- Hydroquinone glucuronide (2): Microsomes were isolated from
cent bioequivalence study between coated tablets containing the liver of 3 starved male Sprague-Dawley rats according to
bearberry leaf extract and the genuine extract [6]. An investiga- methods described previously [9]. 3.26 g of the microsomes
tion on the metabolism of 1 in human volunteers, the renal ex- were resuspended in 5mL 250 mM saccharose solution, and the
cretion of its degradation products, and its further metabolism protein content was determined by the biuret assay. In each of 5
by the bacteria of the uroepithelium was performed by Siegers centrifuge tubes 350 mL suspension of microsomes, 2000 mL 0.5
et al. [7]. In contrast to earlier investigations, a specific HPLC as- M trometamol buffer pH 7.4, 1000 mL 50 mM magnesium chlori-
say for the arbutin elimination products was applied. However, de solution, 500 mL 0.5% Brij 58 solution, 1000 mL 10 mM hydro-
this study as all others apparently determined the total concen- quinone solution and 4150 mL water were mixed. After removal
tration of arbutin metabolites since reference substances of the of air by nitrogen gas, the mixture was pre-incubated with stir-
assumed arbutin metabolites 2 and 6 were not available. A sepa- ring at 378C for 10 min. The synthesis was started by adding
rate determination of 5 and its conjugates was possible by com- 1000 mL 30 mM aqueous uridine 5¢-diphosphoglucuronic acid,
parison of the concentrations of 5 determined in the urine before trisodium salt. After 17 h the reaction was stopped by 20 mL
and after enzymatic hydrolysis of the conjugates with b-glucuro- acetonitrile. The mixture was centrifuged at 850 g for 5 min and
nidase/arylsulfatase (glusulase). With the experimental investi- its volume reduced in vacuum (2 mbar, 308C) to 20 mL. The con-
gations performed for this paper, direct assays for both postu- centrated solution was extracted three times with 20 mL ethyl
lated arbutin metabolites 2 and 6 were published for the first acetate. The aqueous phase was reduced to 5mL. A pH of 2 was
148
time [8].
adjusted with formic acid followed by solid phase extraction
with 75mL 0.05M formic acid. The eluate was evaporated to
The objectives of this paper are the development of an assay of give the crude product as a brown solid substance: HPLC-MS:
the arbutin metabolites 2 and 6 in urine by a HPLC method with m/z = 309 (M + Na)+; UV (DAD): lmax = 283 nm.
fluorometric detection including the synthesis of reference sub-
stances, as far as necessary, and its application to pilot pharma- Methylarbutin (4): Step 1:4-Methoxyphenyl 2,3,4,6-tetra-O-acetyl-
cokinetic investigations of orally administrated bearberry leaf b-D-glucopyranoside (3) was synthesised according to [11]: fine
extracts.
white needles (from ethanol); yield 45%; m.p. 103 8C; anal. calcd.
1
for C21H26O11: C 55.51, H 5.77; found: C 55.41, H 5.83; H-NMR
+
(CDCl3): data according to [11]; MS: m/z = 45 5 (M + H;) UV:
Materials and Methods
Materials
lmax = 224, 282 nm; IR: n = 1746 (C = O), 1509 (C = C), 1229 (C-
OR), 829 cm±1 (doop, = C-H); HPLC purity grade: 99.8%.
The reference substances 1 (Fluka No. 10960; HPLC purity grade Step 2:201 mg (0.44 mmol) 3 were dissolved in 10 mL methanol.
97.7%) and 5 (Fluka No. 53960; HPLC purity grade 98.2%) obtain- After addition of 0.9 mL of a solution of triethylamine in metha-
ed commercially. All reagents used for analytical investigations nol 30% (m/v) and of 5mL (228 mmol) water the mixture was
and for syntheses were of analytical purity. Samples for HPLC stirred for 24 hours at 368C. The solution was evaporated to dry-
were filtered through membrane filters with a mean pore size of ness (368C/20 mbar). Residual triethylamine was removed by re-
0.45 mm (Macherey-Nagel, Düren, Germany). The bearberry leaf peatedly re-dissolving in methanol and evaporation until no
sample was collected in 1997 in Spain (Caelo, Hilden, Germany). odour of triethylamine was perceptible. Re-crystallisation gave
The purification of the enzymatically prepared 2 by solid phase 4:104 mg (0.36 mmol; 37%) white needles (from water); m.p.
extraction was performed with a Bakerbond C18 column (Mal- 177 8C; anal. calcd. for C13H18O7: C 54.54, H 6.34; found: C 54.37,
linckrodt Baker, Griesheim, Germany).
H 6.35; 1H-NMR (CDCl3): data according to [13]; MS: m/z = 287
Quintus J et al. Urinary Excretion of¼ Planta Med 2005; 71: 147±152

(M + H)+; UV: lmax = 284 nm; IR: n = 3392 (O-H), 1511 (C = C), Arbutin metabolites in urine: Urine samples were diluted to a de-
1101 (C-OH), 823 cm-1 (doop, = C-H); HPLC purity grade: 100.0%.
fined volume resulting in concentrations of the analyte within
the controlled linearity range. Blank urine was diluted 1:100.
Hydroquinone sulphate (6): This was synthesised by modification The samples were membrane filtrated and chromatographed
of a published method [10]. 1560 mg (16.6 mmol) phenol were using an aqueous solution of 5(50 ng/mL) as external standard.
dissolved in 50 mL of aqueous potassium hydroxide solution Mobile phase A: 1 mL acetic acid 1%(v/v) + acetonitrile [92.5+ 7.5
10% (m/v), cooled (108C) and the air removed by a continuous ni- (v/v)]; mobile phase B: 1 mL acetic acid 1%(v/v) + acetonitrile [75
trogen stream. A solution of 4580 mg (16.9 mmol) potassium + 25(v/v)]:
persulphate in 100 mL water was added slowly (about 0.6 mL/
min). After 4 hours a pH of 8 was adjusted with carbon dioxide.
The solution was extracted twice with 100 mL of diethyl ether.
The aqueous layer was evaporated to dryness under vacuum,
the residue pulverised and extracted twice with 40 mL ethanol
90% (v/v). The filtrated extract was evaporated to dryness, the
oily product dissolved in boiling absolute ethanol and filtered
immediately. The precipitated product was re-crystallised sever-
al times: 272 mg (1.2 mmol; 7.2%) yellow-white crystals (from
ethanol/petrol ether); m.p. 2328C (decomposition); anal. calcd.
Gradient:
time
[min]
mobile phase A
[%(v/v)]
mobile phase B
[%(v/v)]
0 ± 11
11 ± 14
14 ± 17
17 ± 23
23 ± 35
100
0
100 ® 0
0
0 ® 100
100
0 ® 100
100
100 ® 0
100
for C6H5KO5S: C 31.57, H 2.21, S 14.05; found C 31.40, H 2.18, S Flow: 0.705mL/min; injection volume: 10 mL; fluorescence de-
1
13.46; H NMR (D2O): d = 6.89 (d, J = 8.97, 2H, C-3,5 ), 7.19 (d, tection: lexc = 290 nm, lem = 326 nm; retention times (tR) and
J = 8.98, 2H, C-2,6); MS (neg.): m/z = 189 (M±); UV: lmax
=
correction factors (f) due to different sensitivity: 2: tR = 6.2
231, 276 nm; IR: n = 335 3 (O-H), 1604 (C = C), 15 12 (C = C), min, f = 3.414; 6: tR = 8.5min, f = 11.220; 5: tR = 10.5min. Lin-
n
d
as = 1255 (S-O), 1209 (S-O), ns = 1044 (S-O), n = 840 (S-O-C), earity, LOD and LOQ, calculated to DIN 32645, and precision
oop = 817 (= C-H), d = 587 (SO3), 564 cm±1 (SO3); HPLC purity were performed on urine containing the metabolites 2 and 6
grade: 97.8%.
and spiked with 5 (Tables 3 and 4). A systematic error of 4% re-
sults from the shift of the parallel calibration curves of 5 in the
absence and in the presence of urine. The concentrations of 2
Assays
Aqueous bearberry leaf extract: 1 part of powdered bearberry leaf and 6 in urine did not significantly change within 8 days at
(250) and 100 parts of water were boiled for 10 min and filtered. room temperature without light protection. The robustness of
The filtrate was diluted 1:10 (v/v) with water and chromato- the HPLC assays was also investigated with respect to the chro-
graphed using an aqueous solution of 1 (100 mg/ml) as external matographic conditions. The separation capacity was not sensi-
standard. Mobile phase A: methanol + water [2.5+ 97.5(v/v)];
mobile phase B: methanol + water [15+ 85(v/v)]:
tive to small deviations from the provided polarity of the mobile
phase, but proved to be sensitive to a significant decrease of the
concentration of acetic acid.
149
Gradient:
time
[min]
mobile phase A
[%(v/v)]
mobile phase B
[%(v/v)]
Pilot pharmacokinetic studies
Three healthy volunteers (female, aged 41, weight 53 kg, height
168 cm; male, aged 29, weight 80 kg, height 181 cm; male, aged
60, weight 88 kg, height 173 cm) received each orally 150 mg of 1
and negligible amounts of 4 and 5 in about 150 mL aqueous bear-
berry leaf extract at 8:00 a.m., 2:00 p.m., 8:00 p.m. and 12:00
p.m. Alcohol, caffeine-containing beverages and nicotine were
prohibited during the study. No food was allowed 12 hours prior
0 ± 7.5
7.5 ± 15
15 ± 28
28 ± 30
30 ± 40
100 ® 82
82 ® 0
0
0 ® 18
18 ® 100
100
0 ® 100
100
100 ® 0
100
Flow: 1.5mL/min; injection volume: 15 mL; detection wave- to the study. At 9:30 a.m. two rolls, at 12:00 a.m. a spaghetti
length: 284 nm; retention times (tR) and correction factors (f) meal and from 2:00±6:00 p.m. two other rolls were given. Din-
due to different sensitivity: 1: tR = 4.5min; 5: tR = 6.0 min, ner of one's own choice was allowed after 8:00 p.m. Study ended
f = 0.368; 4: tR = 18.5min, f = 1.073. Linearity, limit of detec-
at 8:00 a.m. the next morning.
tion (LOD) and limit of quantification (LOQ) defined according
to DIN 32645were investigated with 10 aqueous solutions for 1 Urine was collected immediately before the beginning of the
and with 12 aqueous solutions for 4 and 5 with the results listed study, at 10:00 a.m., 12:00 a.m., 2:00 p.m., 4:00 p.m., 6:00
in Table 1. The results of the control of precision of the assays of 1, p.m., 8:00 p.m., 12:00 p.m. and at 8:00 a.m. next morning be-
4 and 5 of the aqueous bearberry extracts and of the accuracy fore breakfast.
tested as recoveries in 50% of the extract amount provided in
the testing procedure spiked with different concentrations of
the analytes are summarised in Table 2. When protected from Results and Discussion
light, solutions of 1, 4 and 5 did not significantly change within
3 days. The separation capacity of the HPLC assays was neither The reference substances which were used as external standards
sensitive to changes of the temperature nor to small deviations for the HPLC assays and not commercially available were bio-
of the flow rate but proved to be sensitive to changes of the com- chemically prepared or synthesised by modified methods de-
position of the mobile phase.
scribed in the literature.
Quintus J et al. Urinary Excretion of¼ Planta Med 2005; 71: 147±152

Table 1 Linearity, LOD and LOQ of the assays of bearberry leaf extract
Analyte
Concentration range
(mg/mL)
Slope
Intercept
R2
LOD (mg/mL)
LOQ (mg/mL)
1
4
5
39.34±393.4
2.036±24.43
0.806± 9.67
2179
2991
7938
±7053
239
0.999960.059
0.99993
0.180
0.070
0.013
0.203
0.041
±1648
0.997
Table 2 Precision and accuracy of the assay of bearberry leaf extract
Analyte
Precision
Concentration
Accuracy
% RSD
Concentration Range
Recovery
% RSD
(mg/mL)
(n = 6)
(mg/mL)
(n = 6)
1
4
5
155
0.98%
2.98%
8.59%
106±203
99.2
97.0
99.9
1.46
1.28
2.38
2.70
3.34
6.36±22.7
3.29±7.73
Table 3 Linearity, LOD and LOQ of the assays of the urine
Analyte
Concentration range
(ng/mL)
Slope
Intercept
R2
LOD
(ng/mL)
LOQ
(ng/mL)
2
5
6
36.5±364.8
9.788±97.88
91.5±915
1486
5003
469
510
3284
0.9999
0.9998
0.9996
2.43
1.297
16.2
7.79
3.954
49.8
22043
150
Hydroquinone glucuronide (2)
ma = 318 nm and 418 nm) were nearly identical with those of
The structure of the main peak of the product which could be the related 1 (UV spectrum: lmax = 284 nm; fluorescence spec-
isolated from the reaction mixture obtained in the biochemical trum: emission maxima = 318 nm and 415nm).
preparation of 2 was identified by UV and fluorescence spectra,
by HPLC-MS experiments [acetic acid in the mobile phase was Methylarbutin (4)
replaced by formic acid and the flow speed was adopted (0.5 The most suitable route for the synthesis of 4 from the different
mL/min)], and by transformation of the glucuronide to its agly- methods described in the literature is the condensation of per-
cone hydroquinone by enzymatic hydrolysis with b-D-glucuroni- acetylated glucose with the aglycone hydroquinone monomethyl
dase (EC 3.2.1.31). The UV and fluorescence spectra of 2 (UV spec- ether in presence of boron trifluoride [11]. The intermediate 3
trum: lmax = 283 nm; fluorescence spectrum: emission maxi- was then hydrolysed to 4 with triethylamine. The structures of
the synthesised products 3 and 4 were confirmed by elemental
analysis as well as by UV, IR, mass, and 1H-NMR spectroscopic
data.
Table 4 Precision of the assays of the urine
Hydroquinone sulphate (6)
Analyte
Precision
Aromatic semi-esters of sulphuric acids like 6 are only known in
the form of their salts [13]. Compound 6 occurs as a non-isolated
intermediate in the synthesis of 5 using a general method for the
preparation of dihydroxybenzenes by oxidation of phenolic com-
pounds with potassium persulphate [10]. Compound 6 was pre-
pared and isolated by a modification of this general synthesis in a
HPLC purity grade of 97.8% from phenol as starting material.
Concentration (ng/mL)
% RSD (n = 6)
2
74.9
76.1
149
1.64
4.10
1.78
1.88
145
6254
1.97
304
3.11
1.54
Assay of the arbutin metabolites
The gradient HPLC with fluorescence detection developed in this
paper enables the perfect separation of the arbutin metabolites
5361.05
590
Quintus J et al. Urinary Excretion of¼ Planta Med 2005; 71: 147±152

Fig. 1 Blank urine (a) and urine obtained
after oral intake of a bearberry leaf extract
additionally spiked with hydroquinone (b).
2, 5 and 6 not only from one another, but also from uric acid and
other urine components (Fig.1). The analytes are separated in
the isocratic part of the chromatogram, the gradient is only ap-
plied in order to eliminate components of urine which would
challenge the column and result in a limitation of column capa-
city. While UV detection gives a very complicated chromatogram
due to the numerous urine components, the applied fluorometric
detection (excitation: 290 nm; emission: 326 nm) permits a
specific determination of the three mentioned metabolites, par-
ticularly without interference with uric acid which gives a very
strong peak under UV detection.
Metabolites 5 and 6 are available as pure reference substances,
and thus an experimentally determined response factor can be
used for the calculation of their concentrations. The concentra-
tion of the metabolite 2 which could not be isolated in pure
form is calculated related to 1 considering a stoichometric cor-
rection factor. Identical detection sensitivity related to molar
concentrations of the analyte and the external standard may be
assumed due to their identical fluorophores.
151
Pharmacokinetic results
In a preliminary pharmacokinetic study the time-dependent ex-
cretion of arbutin metabolites after multiple administration of
an aqueous bearberry leaf extract containing 150 mg of arbutin
per single dose was investigated on 3 healthy volunteers. The
dosage complies with the recommendations of the Kommission
E [14] responsible for the evaluation of herbal remedies in Ger-
many. This monograph recommends a daily intake of 400±
840 mg hydroquinone derivatives, calculated as arbutin, divided
into up to 4 single doses of each 100±210 mg. The single doses
were repeated 6,12, and 16 h after the first dosage. Urine samples
were collected immediately before and 2, 4, 6, 8, 10, 12, 16, and
24 h after the first application, and the concentrations of the ar-
butin metabolites were determined by HPLC. The arbutin meta-
bolites in the blank urine fraction were additionally determined
since hydroquinone derivatives occurring in different foods and
tobacco smoke [15] can also be transformed to the determined
metabolites. In the blank urines of the three volunteers collected
immediately before application concentrations of the metabo-
lites 6 and 7 were below the detection limit. Compound 2 was Fig. 2 Urinary elimination profiles of arbutin metabolites
Quintus J et al. Urinary Excretion of¼ Planta Med 2005; 71: 147±152

Table 5 Renal elimination of arbutin metabolites
Total excretion of test person
2
1
3
4 h after first intake of 150 mg arbutin,
Calculated as arbutin [mg]
81.7
54.5
475
79.3
94.1
62.7
593
88.4
58.9
574
95.7
[%] of the applied arbutin
24 h after first intake, total amount of 600 mg arbutin, calculated as arbutin [mg]
[%] of the applied arbutin
99.0
only found in urine of one volunteer at a concentration of References
6.71 mg/mL.
1
Hänsel R, Haas H. Therapie mit Phytopharmaka. Berlin: Springer Ver-
lag, 1983: p 197
The urinary elimination profiles of the arbutin metabolites 2, 5
and 6 after oral intake of the bearberry leaf extract are graphical-
ly summarised in Fig. 2. The renal elimination of 2 and 6 already
began within the first 2 h, and decreased until the second intake
after 6 h. More than half of the administered dose of arbutin was
excreted within 4 h and more than 75% of the total applied arbutin
within 24 h (Table 5). The concentration of 5 in urine of volun-
teers 1 and 2 was above the limit of quantitation in only one frac-
tion (3±4 and 13±16 hours after the first application) where the
elimination did not exceed 0.130 mg of 5, corresponding to
0.3 mg arbutin. The third volunteer excreted 1.53 mg of 5, cor-
responding to 3.77 mg arbutin and to 2.5% of the administered
dose of 150 mg arbutin within the first dosage interval of 6 h.
This test person excreted 13.7 mg of 5, corresponding to 33.9 mg
arbutin and to 5.6% of the total applied dose of 600 mg arbutin
within 24 hours. Siegers et al. [7] could also detect 5 in urine
samples in 6 out of 12 test persons. The mean amount of renally
eliminated 5 of all 12 test persons corresponded to 0.6% of the
administered arbutin dose.
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The observed profiles of renally excreted arbutin metabolites
confirm the meaningfulness of the mentioned recommendation
of the German Kommission E to administer the provided dose of
immediate release preparations of bearberry leaf divided into 4
single doses. In accordance with previous results [5], [6], [7],
but in contrast to the results of [16] the occurrence of only one
main metabolite of arbutin in urine cannot be confirmed.
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Acknowledgements
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Schindler G, Patzak U, Brinkhaus B, von Nieciecki A, Wittig J, Kraehmer
N, Veit M. Urinary excretion and metabolism of arbutin after oral ad-
ministration of Arctostaphylos uvae ursi extracts as film-coated tablets
and aqueous solution in healthy humans. J Clin Pharmacol 2002; 42:
920±7
We thank Prof. J. Werringloer for the isolation of rat liver micro-
somes and Prof. K. Albert and his group for assistance in struc-
ture elucidation by LC-MS investigations.
Quintus J et al. Urinary Excretion of¼ Planta Med 2005; 71: 147±152