Prevention of Experimentally Induced Irritant Contact Dermatitis by Extracts of Isatis tinctoria Compared to Pure Tryptanthrin and its Impact on UVB-Induced Erythema

• Abstract
• Introduction
• Material and Methods
• Plant material, preparation of extracts and test solutions
• Study design
• Statistical evaluation
• Results
• Discussion
• Acknowledgements
• References

Abstract

Lipophilic extracts of Isatis tinctoria L. exhibit significant activity against several clinically relevant targets of inflammation. The alkaloid tryptanthrin was identified as one of the active principles in woad and characterised as a potent dual inhibitor of COX-2 and 5-LOX. Here, the anti-inflammatory efficacy of topical application of three different Isatis extracts and tryptanthrin was investigated in human volunteers. Two different models were used, namely the sodium lauryl sulphate (SLS)-induced irritant contact dermatitis (ICD) and UVB-induced erythema. Twenty healthy volunteers without any skin disease participated in the study. Cumulative irritant contact dermatitis was induced on test fields on the volunteers’ backs by twice daily application of 0.5 % sodium lauryl sulphate over a period of four days. Half of the test fields were treated with the test substances during the eliciting phase, while the remaining test fields were treated over a period of 4 days after induction of dermatitis. In the second model, a UVB erythema on the volunteers’ lower backs was induced using the double minimal erythema dose (MED). Twenty-four hours after irradiation the test fields were treated with the test substances over a period of 3 days. All reactions were assessed visually and by non-invasive bioengineering methods (evaporimetry and chromametry). Treatment with extracts during the ICD eliciting phase led to a significantly smaller increase of visual scores and transepidermal water loss compared to the untreated test field. For tryptanthrin this benefit was also observed, but the improvement was not statistically significant. When treatment was performed after completing the eliciting phase, accelerated resolution of the irritant reaction could not be observed. In the UVB erythema model anti-inflammatory effects of the test substances were not observed.

Introduction

A wide range of anti-inflammatory substances is available for the treatment of inflammatory skin diseases. However, most of them (e. g., corticosteroids) have considerable side effects [1]. Therefore, the interest in new substances for the treatment of such skin diseases remains high. Due to the high acceptance of herbal drugs among the population, plant-based preparations with demonstrated clinical efficacy could be a suitable alternative. Herbs such as marigold (Calendula officinalis L.) and chamomile [Matricaria recutita (L.) Rauschert] are currently used in phytotherapy as topical anti-inflammatories. Although their use is supported by pharmacological evidence, the mode of action largely remains open to conjecture [2].

On the basis of a survey of historical accounts on old folk medicines with potential anti-inflammatory activity followed by in vitro pharmacological screening, we recently identified the ancient indigo dye [3], and medicinal herb Isatis tinctoria L. (woad, family Brassicaceae) [4] as an anti-inflammatory of considerable interest. Lipophilic Isatis extracts displayed a promising in vitro profile against a range of clinically relevant targets, including inhibition of cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), and of histamine and serotonin release from mast cells [5]. Subsequently, the indolo[2,1-b]quinazoline alkaloid tryptanthrin (1) (Fig. [1]) was identified as one of the active principles in woad and characterised as a potent dual inhibitor of COX-2 and 5-LOX [6], [7]. The anti-inflammatory potential of Isatis extracts and tryptanthrin was further supported in phorbol ester-induced mouse ear oedema and in carrageenan-induced paw oedema as models for acute inflammation [5]. Interestingly, the activity of the extracts was higher than that of tryptanthrin (1), suggesting synergistic effects of multiple active principles or penetration enhancement by extract constituents. Skin penetration of tryptanthrin (1) upon topical application was recently demonstrated by skin microdialysis [8]. However, in spite of the long tradition of medical use of woad extracts, no studies have been published so far to provide evidence of their clinical effectiveness in topical applications. In this study we investigated the anti-inflammatory efficacy of three different Isatis extracts and pure tryptanthrin in sodium lauryl sulphate (SLS)-induced irritant contact dermatitis (ICD) and UVB-induced erythema.

Material and Methods

Plant material, preparation of extracts and test solutions

Leaf material was harvested from first year plants of a defined Isatis tinctoria L. culture, ”Thüringer Waid”, grown on experimental plots of the Agricultural Research Station of Thuringia (TLL), Dornburg, Germany. A voucher specimen (ISAR01) is preserved in the Herbarium of the Institute of Pharmacy, University of Jena. Fresh leaves were dried on a band drier operating at 60 °C. Extracts (E1 - E3) were prepared at the Adalbert-Raps-Zentrum, Technical University München-Weihenstephan, in a pilot plant supercritical fluid extractor. Dried leaves were extracted for 3 h at 50 °C with CO₂ or CO₂-EtOH at varying pressures. The extraction conditions and yields were as follows: E1: CO₂ at 800 bar, yield 0.8 %; E2: CO₂ + 5000 ppm EtOH at 350 bar, yield 1.2 %; E3: CO₂ + 5000 ppm EtOH at 700 bar, yield 1.9 %. The tryptanthrin contents of the extracts, determined according to the procedure by Danz et al. [9], were 0.174 % for E1, 0.209 % for E2, and 0.103 % for E3, respectively. Solutions of extracts (5 % in acetone) were prepared for testing. Tryptanthrin was synthesised [10] and characterised by m. p., UV/VIS, HPLC/MS, IR and NMR. Purity was ≥ 99 %.

For testing, acetone solutions were prepared from extracts (5 %) and from tryptanthrin (1 %). For control, acetone without test substance was applied onto separate test fields. All substances were applied onto test areas on the back according to a randomisation scheme.

Study design

The study was designed and performed in a randomised single-blinded manner under standardised laboratory conditions between January and May 2001. Twenty healthy volunteers [15 women, 5 men; mean age 26.7 (± 6.9) years; range 18 - 44 years] without any skin disease participated in this study after giving written informed consent in accordance to the Helsinki declaration. During the 10 days of investigation the volunteers were allowed to shower as usual but they had to avoid bathing, application of detergents, moisturisers or emollients on their backs. They were also asked to avoid sun beds and solar radiation. The study had passed review by the Ethical Commission of the Friedrich-Schiller University.

The skin irritation was provoked by sodium lauryl sulphate. Tests were performed from Monday through Thursday at the same (± 1 h) time of day. Eleven fields on the paravertebral skin of the back were used as test areas. The cumulative irritation was induced by occlusive application of 50 μL of 0.5 % sodium lauryl sulphate (99 %, Serva Feinbiochemie, Heidelberg, Germany) dissolved in distilled water. The irritant was applied by using large Finn chambers on Scanpor (Ø 12 mm) (filling volume 50 μL, Epitest, Hyrla, Finland) with filter discs which were transferred twice daily with 3 h intervals to the skin [11]. After 30 minutes of exposure the Finn chambers were removed and the skin was dried carefully using paper tissues.

To effect a UVB erythema the minimal erythema dose (minimal dose which provokes erythema in a certain individual, MED) was determined with increasing UVB doses (0.05; 0.075; 0.1; 0.125; 0.150; 0.175 J/cm²) in six 1 cm² gluteal fields. Irradiation was performed with the Waldmann UV800 irradiator (Herbert Waldmann GmbH, Villingen-Schwenningen, Germany; emission spectrum: 285 - 350 nm; intensity: 3.12 mW/cm²). Visual reading was performed after 24 h [12]. Thereafter, 6 additional fields were irradiated with the double MED.

After exposure to the irritant or after irradiation, respectively, the test preparations (50 μL of extracts: E1 : 4.4 μg, E2 : 5.2 μg, E3 : 2.6 μg tryptanthrin; pure tryptanthrin solution: > 50 μg tryptanthrin) were pipetted into glass O-rings (inner diameter 12 mm, height 15 mm; QVF-Labortechnik, Ilmenau, Germany) which were pressed onto the test areas until evaporation of the solvent. In 5 test fields the test substances were applied ten minutes after SLS irritation (parallel treatment). Five days later, the final measurements were made in these fields.

In 5 additional fields on the back, application of the solutions started on the fourth day, 10 minutes after the last SLS exposure. Application was performed once daily at the same time (± 1 h) for a total of 5 times until day 8. Another field served as control with irritation only.

In the 6 gluteal fields with UVB-induced erythema, treatment was started 24 h after irradiation and continued once daily for 2 more days. The resolution of the erythema was compared to a control field which was irradiated only.

To assess possible irritant effects of the test substances themselves and to investigate their influence onto the chromametric a* values due to skin staining, 4 additional fields in the back were treated twice daily (with 3 h intervals) for 4 days with the test substances only. Measurements were made on day 1 and day 5.

The biophysical measurements started after the volunteers had rested for at least 15 min and after acclimatisation to the standardised laboratory conditions (temperature 20 - 22 °C, humidity 20 to 40 %). They were made by the same observer at the same time (± 1 h), 10 min before irritation or, respectively, irradiation. From day 5 on, only the test substances were applied, and the measurements were conducted as before. The final measurements were performed on day 9, 24 h after the last application.

The following methods were used: Visual scoring was performed according to Frosch and Kligman [13]. Transepidermal water loss (TEWL) as an indicator of epidermal barrier function was measured using the Tewameter TM 210® (Courage & Khazaka, Cologne, Germany) and followed the guidelines of Pinnagoda et al. [14]. The intensity of erythema was quantified by using the chromameter CR-300® (Minolta GmbH, Ahrensburg, Germany) according to the recommendations of Elsner [15] by exclusively considering the a* value which represents the red-green axis of the three-dimensional L*-a*-b*-assay system.

Positive controls were not included in the study protocol, since there is no golden standard for the treatment of irritant contact dermatitis. Especially, there is no evidence base for the use of topical corticosteroids in the treatment of irritant contact dermatitis [16].

Statistical evaluation

Statistical calculations were carried out with the SPSS 10.0 for Windows software package. For each test field, the mean values of the chromametry and TEWL data points were calculated from three and two measurements, respectively. The comparisons of the test and control data were performed with the non-parametric Wilcoxon test. Differences with a probability of error of less than 5 % were regarded as significant. Corresponding to the amount of paired comparisons, levels of significance were adjusted according to Bonferroni.

Results

Application of the Isatis extracts onto untreated test fields (twice daily for 4 days) led to a brown-yellow staining of the skin. However, the visual score remained at 0 during the period of observation. Changes in TEWL were not significant (data not shown). The chromametric a* values showed a significant increase from day 1 to day 5 (extract 1: p ≤ 0.05; extracts 2 and 3: p ≤ 0.01). Tryptanthrin in acetone did not have a significant influence on chromametry (Fig. [2]).

After treatment with Isatis tinctoria extracts (E1 - E3) the visual score (on test fields which were treated during the induction phase of the SLS dermatitis) increased to a significantly smaller degree compared to the exclusively SLS-treated control field (E1 and 2: p ≤ 0.001; E3: p ≤ 0.01). Tryptanthrin alone did not influence the visual score significantly. TEWL increased to a significantly smaller degree under treatment with the Isatis extracts, too. TEWL values on the tryptanthrin-treated test field were not significantly smaller than on the untreated control field (E1, 2 and 3: p ≤ 0.001; tryptanthrin: p ≤ 0.05). As measured by chromametry, the erythema on test fields under application of the extracts 1 and 2 was significantly reduced compared to the control field (p ≤ 0.01). For extract 3 and tryptanthrin this difference was not significant. Acetone enhanced irritation slightly but without reaching significance in comparison to control (Figs. [3] [4] [5]).

When the test substances were applied after the end of the SLS irritation period, an accelerated resolution of the irritant reaction could not be observed with any method (Fig. [6], data of the visual scoring and chromametry not shown).

The visual score failed because the degree of the erythema of treated test fields was indiscernible by eye from that of the control sites. In spite of a considerable erythema after UVB irradiation, the TEWL did not markedly increase. Therefore, the reduction of the TEWL over the 4 days of measurement did not differ significantly between the treated sites and the untreated control site. Chromametry revealed a slight effect of the extracts on the erythema resolution between 24 h and 48 h after irradiation. However, this was not statistically significant (Fig. [7]).

Discussion

To our knowledge, this is the first study on the topical anti-inflammatory effect of Isatis tinctoria extracts and tryptanthrin in humans. We showed that the extracts and tryptanthrin caused no irritant reaction on healthy skin. Chromametric a* values were significantly augmented due to the skin coloration by the extracts, a fact that has to be considered when chromametry results of treated sites and untreated control sites are compared. However, though statistically significant, the difference was rather small.

When application of the extracts was performed during the induction phase of SLS-induced ICD (parallel treatment), the induced irritant reaction at day 5 was significantly lower than on the untreated, irritated control field. Tryptanthrin also showed an obvious tendency to reduce the development of inflammation. However, after performing the adjustment of the p value according to Bonferroni, the difference to the untreated control site was no longer statistically significant. The better efficacy of Isatis extracts in these skin irritation models is corroborated by the in vivo pharmacological data in the phorbol ester-induced mouse ear oedema, where significant anti-oedemateous effects were observed upon topical application of extracts (0.5 mg/ear) but not with pure tryptanthrin at 0.25 mg/ear [5]. Different reasons may be responsible for the better efficacy of Isatis extracts: One is that compounds other than tryptanthrin appear to contribute to the anti-inflammatory activity. Indeed, the in vitro pharmacological profile of Isatis extracts shows that their activity cannot be solely explained by tryptanthrin [5]. A thorough analysis of other active principles in Isatis extracts is underway. Secondly, we were able to show by skin microdialysis that penetration of tryptanthrin is higher from extracts than from solutions of pure compound. The alkaloid has a tendency to crystallise on the skin surface after application of a pure tryptanthrin solution but remains in molecular dispersion in the extract [8]. In addition, the skin penetration of anti-inflammatory substances may be enhanced by other extract constituents [17].

When treatment was started just after induction of ICD, no differences could be found in the recovery compared to the untreated control field. The results suggest that application of the extracts is more effective in the developmental stages of inflammation. Tryptanthrin was recently shown to inhibit cyclooxygenase-2 and 5-lipoxygenase in vitro [7]. In early inflammation the expression of the COX-2 gene is up-regulated and pro-inflammatory metabolites derived from arachidonic acid are generated [18]. In a suction blister study, increased amounts of prostaglandins and other eicosanoids were found in SLS-induced ICD [19]. Probably, inhibition of COX-2 is more likely to prevent the progression of inflammation than accelerating recovery from barrier damage. UVB irradiation of the skin also causes up-regulation of the COX-2 gene expression [20], [21]. We therefore supposed the UVB erythema model to be a good tool to study the efficacy of COX-2-enzyme inhibitors as tryptanthrin. However, TEWL values were only influenced to such a small extent by irradiation with the double MED that this parameter was not suited to secure a possible positive effect of the test substances. The effect of the test substances on chromametry values was statistically not significant.

Summarising our findings, Isatis tinctoria extracts were effective in the prevention of ICD in the eliciting phase. The benefit of pure tryptanthrin was small and statistically not significant. In order to use Isatis extracts in the treatment of inflammatory skin diseases, an appropriate galenic formulation will be necessary to guarantee optimal release and penetration into the skin. Potentially irritant solvents like acetone which had to be chosen in this pilot study due to the poor solubility of tryptanthrin should be avoided. Given the much better solubility properties of Isatis extracts, this goal appears achievable without major difficulties.

Acknowledgements

Dr. A. Vetter and Mrs. A. Biertümpfel, Agricultural Research Station of Thuringia, Dornburg, are gratefully acknowledged for the provision of plant material. We thank Dr. B. Weinreich, Adalbert-Raps-Forschungszentrum, TU München-Weihenstephan, for preparation of the SFE extracts.

References

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  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar

Prof. Dr. Peter Elsner

Department of Dermatology and Dermatological Allergology

University of Jena

Erfurter Str. 35

07740 Jena

Germany

Phone: +49-3641-937-370

Fax: +49-3641-937-343

Email: elsner@derma.uni-jena.de

References

• 1 Thiers B H. Topical steroid therapy of atopic skin diseases.  Allergy Proc. 1989;  10 413-6
  PubMedSearch in Google Scholar
• 2 Wichtl M. Teedrogen und Phytopharmaka. 4th Edition Stuttgart; Wissenschaftliche Verlagsgesellschaft 2002
  Search in Google Scholar
• 3 Hurry J B. The woad plant and its dye. London; Oxford University Press 1930
  Search in Google Scholar
• 4 Tang W, Eisenbrand G. Chinese drugs of Plant Origin. Berlin; Springer-Verlag 1992
  Search in Google Scholar
• 5 Hamburger M. Isatis tinctoria - from the rediscovery of an ancient medicinal plant towards a novel anti-inflammatory phytopharmaceutical.  Phytochem Rev. 2002;  1 333-44
  CrossrefPubMedSearch in Google Scholar
• 6 Danz H, Stoyanova S, Wippich P, Brattström A, Hamburger M. Identification and isolation of the cyclooxygenase-2 inhibitory principle in Isatis tinctoria .  Planta Med. 2001;  67 411-6
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Danz H, Stoyanova S, Thomet O AR, Simon H U, Dannhardt G, Ulbrich H. et al . Inhibitory activity of tryptanthrin on cyclooxygenases-1 and -2, and 5-lipoxygenase.  Planta Med. 2002;  68 875-80
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Oberthür C, Heinemann C, Elsner P, Benfeldt E, Hamburger M. A comparative study on the skin penetration of pure tryptanthrin and tryptanthrin in Isatis tinctoria extract by subcutaneous microdialysis coupled with isotope dilution ESI-LC-MS.  Planta Med. 2003;  69 385-9
  Thieme ConnectPubMedSearch in Google Scholar
• 9 Danz H, Baumann D, Hamburger M. Quantitative determination of the dual COX-2/5-LOX inhibitor tryptanthrin in Isatis tinctoria by ESI-L-MS.  Planta Med. 2002;  68 152-7
  Thieme ConnectPubMedSearch in Google Scholar
• 10 Friedländer P, Roschdestwensky N. Über ein Oxidationsprodukt des Indigoblaus.  Ber dtsch chem Ges. 1915;  48 1841-7
  CrossrefPubMedSearch in Google Scholar
• 11 Wigger-Alberti W, Krebs A, Elsner P. Experimental irritant contact dermatitis due to cumulative epicutaneous exposure to sodium lauryl sulphate and toluene: single and concurrent application.  Br J Dermatol. 2000;  143 551-6
  CrossrefPubMedSearch in Google Scholar
• 12 Bangha E, Elsner P, Kistler G S. Suppression of UV-induced erythema by topical treatment with melatonin (N-acetyl-5-methoxytryptamine). Influence of the application time point.  Dermatology. 1997;  195 248-52
  PubMedSearch in Google Scholar
• 13 Frosch P J, Kligman A M. The soap chamber test. A new method for assessing the irritancy of soaps.  J Am Acad Dermatol. 1979;  1 35-41
  PubMedSearch in Google Scholar
• 14 Pinnagoda J, Tupker R A, Agner T, Serup J. Guidelines for transepidermal water loss (TEWL) measurement. A report from the Standardization Group of the European Society of Contact Dermatitis.  Contact Dermatitis. 1990;  22 164-78
  CrossrefPubMedSearch in Google Scholar
• 15 Elsner P. Chromametry. In: Handbooks of Skinbioengeneering. Cutaneous blood flow and Erythema. (Berardesca E., Elsner P., Maibach H., eds) Boca Raton; CRC Press 1994: pp 247-52
  Search in Google Scholar
• 16 Van der Valk P G, Maibach H I. Do topical corticosteroids modulate skin irritation in human beings? Assessment by transepidermal water loss and visual scoring.  J Am Acad Dermatol. 1989;  21 (3Pt1) 519-22
  PubMedSearch in Google Scholar
• 17 Eder M, Mehnert W. Bedeutung pflanzlicher Begleitstoffe in Extrak7ten.  Pharmazie. 1998;  53 285-93
  PubMedSearch in Google Scholar
• 18 Crofford L J. COX-1 and COX-2 tissue expression: implications and predictions.  J Rheumatol. 1997;  24 (Suppl 21) 15-9
  PubMedSearch in Google Scholar
• 19 Muller-Decker K, Heinzelmann T, Furstenberger G, Kecskes A, Lehmann W D, Marks F. Arachidonic acid metabolism in primary irritant dermatitis produced by patch testing of human skin with surfactants.  Toxicol Appl Pharmacol. 1998;  153 59-67
  CrossrefPubMedSearch in Google Scholar
• 20 Wilgus T A, Ross M S, Parrett M L, Oberyszyn T M. Topical application of a selective cyclooxygenase inhibitor suppresses UVB mediated cutaneous inflammation.  Prostaglandins Other Lipid Mediat. 2000;  62 367-84
  CrossrefPubMedSearch in Google Scholar
• 21 Camp R D, Greaves M W, Hensby C N, Plummer N A, Warin A P. Irradiation of human skin by short wavelength ultraviolet radiation (100 - 290 nm) (u. v.C): increased concentrations of arachidonic acid and prostaglandins E2 and F2-α.  Br J Clin Pharmacol. 1978;  6 145-8
  PubMedSearch in Google Scholar

Prof. Dr. Peter Elsner

Department of Dermatology and Dermatological Allergology

University of Jena

Erfurter Str. 35

07740 Jena

Germany

Phone: +49-3641-937-370

Fax: +49-3641-937-343

Email: elsner@derma.uni-jena.de