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Bacterial deconjugation of Arbutin by Escherichia coli. (Short Communication).

Summary

Cystinol[R] akut containing arbutin was developed as an antiseptic since it liberates hydroquinone in the urine. The in vivo release of hydroquinone from arbutin with or without addition of glusulase or an E.coli suspension was investigated in 4 volunteers. They ingested 6 dragees Cystinol[R] akut (420 mg arbutin equivalent to 168 mg hydroquinone), urine was sampled and assayed by a validated HPLC method. Results: In comparison to incubation with glusulase the E. coli-sus pension resulted in a 2.3fold higher increase in free hydroquinone. When separating bacteria from the urine, the hydroquinone concentration in bacteria was 20fold higher than in the supernatant. Consequences: Glucuronic acid or sulfuric acid conjugates of hydroquinones obviously are taken up, enriched and metabolized to hydroquinone by bacteria. Deconjugation of hydroquinone likely is catalyzed by intracellular enzymes presumably present in bacterial cytoplasm; comparable activities in eucaryotic cells usually are localized in lysosomes. Alk alization of the urine seems not to be a prerequisite to improve the antiseptic properties of hydroquinone released from arbutin.

Key words: Arbutin, Deconjugation, Escherichia coli, hydroquinone

Introduction

The monograph "Uvae ursi folium" of the former german BGA (Anonymus, 1994) recommends alkalisation of the urine in connection with the intake of pharmaceutical formulations of Uvae ursi folium or arbutin. This should enhance the urinary concentration of free hydroquinones released from arbutin and thereby increase the antiseptic effect of these teas or phytopharmaceutical drug preparations. This recommendation was not based on pharmacokinetic studies of the metabolic fate of arbutin in humans or experimental animals. From our previous toxicokinetic study in volunteers (Siegers et al. 1997 a, b) we found that arbutin is metabolized in the human intestinal wall and excreted by the urine mainly as hydroquinone conjugates with glucuronic or sulfuric acid. Less than 0.5% of an orally applied dose are excreted as free hydroquinone, which is likely responsible for the antiseptic effect. The present investigation should clarify whether bacteria in urinary infections participate in the deconjugation of hydroquinone an d liberate the toxic free hydroquinone, thereby killing themselves. This could be interpreted as a suicidal mechanism.

Methods

4 volunteers, aged between 25 und 45 years (2 males/2 females) ingested the recommended daily dose of three times 2 dragees of the drug Cystinol[R] akut corresponding to a total dose of 420 mg arbutin = 160 mg hydro quinone, together with 100 ml mineralwater. The food intake (breakfast, lunch and dinner) was standardized, only vegetarian food was allowed to increase the urinary pH values to more than 6,5. The ethical agreement was given by the committee of the regional "Arztekammer" (Stud.-No. SB-SYS-1094). A commercially available enzyme solution of glusulase (Boehringer, Mannheim) to cleave conjugates in vitro, or a E. coli suspension (ATCC6538) to mimick infection conditions were added to defined urine samples and incubated for 24 hours at 37 [degrees]C in an incubator.

For differentiation between bacterial supernatant and bacterial sediment, 100 [micro]l/ml perchloric acid (333 mmol/l stock solution) was added to precipitate protein. After centrifugation at 4000 rpm (4 [degrees]C the supernatant was carefully decanted and the sediments were weighed.

* Results and Discussion

The comparison of HPLC traces from urinary samples without or with addition of the enzyme glusulase ([beta]-glucuronidase/arylsulfatase) or a suspension of << [10.sup.6] CFU/ml>> E. coli resulted in a 2,3 fold higher concentration of deconjugated free hydroquinone (Tables 1+2, Fig. 1). For both enzymatic cleavage and bacterial activity a comparable increase in the amount of free hydroquinone was observed.

After centrifugation of the samples and separation of the bacteria from the supernatant a 20 fold higher amount of free hydroquinones was detected in the bacterial sediments as compared to the supernatant (Table 3, Fig. 2).

This result indicates uptake and enrichment of hydroquinone conjugates and subsequent liberation of hydroquinone in E. coli bacteria.

The deconjugating enzymes, e.g. [beta]-glucuronidases, are found in the bacteria (Davis et al. 1969; Burrows, 1973), which enrich and deconjugate hydroquinone glucuronides and/or sulfates. The free hydroquinone then can damage the cell by destabilisation of its membranes.

Alkalisation of the urine by intake of bicarbonate, as recommended in the monograph (Anonymus, 1994) to increase the amount of free hydroquinone as the antiseptic principle of uvae ursi-extracts is thus not necessary considering the effective bacterial deconjugation by E. coli, the principal agent in urinary infections.

The therapeutic effect of Cystinol[R] akut can be obtained without alkalisation of the urine. Since the intracellular pH value of E. ccli is not affected by urine alkalisation; additional intake of bicarbonate probably has no influence on the intracellular bacterial deconjugation of hydroquinone conjugates.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]
Table 1

Total hydroquinone in the 24-h urinary samples after incubation with
glusulase (200 [micro]l).

Proband Concentration Urinary Total
 [micro]g/ml volumina amount
 ml/24h mg/24h

1 38.06 1000 38.06
2 19.68 1570 31.02
3 7.04 1260 8.90
4 38.86 1400 54.40
Arithm. 25.91 1307 33.14
mean
S.E.M. 7.69 20 9.43

Table 2

Total hydroquinone in the 24-h urinary samples after incubation with E.
coli ([10.sup.5]/100 [micro]l) suspension.

Proband Urinary Total
 Concentration volumina amount
 [micro]g/ml ml/24 h mg/24 h

1 72.42 1000 72.42
2 47.86 1570 75.14
3 41.95 1260 52.85
4 76.10 1400 106.54
Arithm. 59.58 1307 76.74
mean
S.E.M. 8.59 20 11.11

Table 3

Free hydroquinone concentration after incubation with E. coli suspension
([10.sup.5]/100 [micro]l) in the supernatant and bacterial sediment.

Proband Supernatant Sediment
 [micro]g/ml [micro]g/g

1 65.90 1304
2 46.46 280
3 37.14 962
4 67.42 1736
Arithm. mean 54.23 1070
S.E.M. 7.43 307


* References

Anonymus (1994) Bundesanzeiger 109: Monograph: Uvae ursi folium (Barentraubenblatter). Bundesanzeiger 109 vom 15.06.1994

Burrows W (1973) Textbook of Microbiology. W.B. Sanders Company, pp 37-38

Davis BD, Dulbecco R, Eisen HN, Ginsberg HS, Wood jr. WB (1969) Microbiology. Harper Int. Edition p. 31

Siegers C-P, Pentz R, Siegers J-P (1997a) Oral arbutin in man, results of a toxicokinetic study in man. Naunyn, Schmiedeberg's Arch Pharmacol Suppl 355, R 137

Siegers C-P, Siegers J-P, Pentz R, Bodinet C, Freudenstein J (1997b) Metabolism of arbutin from uvae ursi-extracts in humans. Pharm Pharmacol Lett 7: 90-92

Claudia Siegers (1)

Cornelia Bodinet (2)

Sawar Syed Ali (1)

Claus-Peter Siegers (1)

(1.) Institute of Experimental and Clinical Pharmacology and Toxicology, University of Luebeck, Luebeck, Germany

(2.) Schaper & Bruemmer, Salzgitter-Ringelheim, Germany

Address

C.-P. Siegers, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Luebeck, Ratzeburger Allee 160, D - 23538 Luebeck, Germany

Tel.: ++49-451-500-2697; Fax: ++49-451-500-2303;

e-mail: siegers@medinf.mu-luebeck.de
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Article Details
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Author:Siegers, Claudia; Bodinet, Cornelia; Ali, Sawar Syed; Siegers, Claus-Peter
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Product/Service Evaluation
Geographic Code:4EUGE
Date:May 1, 2003
Words:1140
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