Catechol, a Bioactive Degradation Product of Salicortin, Reduces TNF- α ...
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Catechol, a Bioactive Degradation Product of Salicortin, Reduces TNF-α Induced ICAM-1 Expression in Human Endothelial Cells Susanne Knuth 1 , Helmut Schübel 2 , Martin Hellemann 2 , Guido Jürgenliemk 1 1 Department of Pharmaceutical Biology, Institute of Pharmacy, University of Regensburg, Regensburg, Germany 2 Hermes Arzneimittel GmbH, Großhesselohe/Munich, Germany Abstract ! The phenolic glucoside salicortin was isolated from a Willow bark extract, and its ability to reduce the TNF-α induced ICAM-1 expression (10 ng/mL, 30 min pretreatment with salicortin) was tested in vitro on human microvascular endothelial cells (HMEC- 1). After 24 h, 25 μM salicortin decreased the TNF-α induced ICAM-1 expression to 65.9% compared to cells which were treat- ed only with TNF-α. In parallel, the stability of 25 μM salicortin under assay conditions was determined by HPLC. Within 24 h, the salicortin concentration decreased to 3.1 μM whereas cate- chol, a known NF-κB inhibitor, rose as a metabolite. After 8 h the catechol concentration was relatively constant and varied be- tween 8.2 and 10.9 μM. Considering this degradation in the in vi- tro test system, 10 μM catechol was added 8 h after TNF-α stimu- lation, and 16 h later the ICAM-1 expression was determined. In this setting, the ICAM-1 expression was reduced to 74.8%. This is comparable to the effect obtained from 25 μM salicortin and indi- cates that its activity is related to the generation of catechol, as salicin, saligenin, and salicylic acid are only marginally active or inactive in this test system in a concentration up to 50 μM. These results indicate catechol as an important bioactive metabolite from salicortin. Key words Salix · Salicaceae · salicortin · catechol Abbreviations ! TNF-α: tumor necrosis factor α ICAM-1: intercellular adhesion molecule 1 NF-κB: nuclear factor-κB HMEC-1: human microvascular endothelial cells Supporting information available online at http://www.thieme-connect.de/ejournals/toc/plantamedica Herbal remedies containing willow bark extract (Salicis cortex, Salix spec., Salicaceae) are used since ancient times to treat fever, pain, and inflammation [1]. Although salicylic acid, as the in vivo metabolite of salicylic alcohol derivatives, was described as the active principle of willow bark, several studies pointed out that other ingredients like polyphenols contribute to the overall effect of the drug due to the low achievable salicylic acid plasma con- centration [2,3]. To find out if the salicylic alcohol derivatives themselves could contribute to the anti-inflammatory activity via hitherto not investigated mechanisms, salicortin was isolated from a willow bark extract, and its ability to reduce the TNF-α in- duced ICAM-1 expression in endothelial cells was investigated. The stability of salicortin in cell culture medium was determined by HPLC. Salicortin was isolated from a willow bark extract by column chromatography with Sephadex-LH20 ® , RP-18, and silica gel (l " Fig. 1). In an in vitro assay with human microvascular endo- thelial cells (HMEC-1), 10 to 75μM salicortin reduced the TNF-α induced ICAM-1 expression dose-dependently after 24 h of incu- bation (30 min pretreatment with test substances; l " Fig. 2). In these concentrations no influence on the cell viability was ob- served as indicated by MTT assays. Interestingly, 50 μM of salicin, salicylic acid, and saligenin showed no or only slight effects (l " Fig. 2), whereas the same concentration of salicortin reduced the ICAM-1 expression to 52.4 %. Ruuhola et al. analyzed the in vitro degradation of salicylates under different pH values and determined the influence of differ- ent enzymes. Results revealed that salicortin is unstable at pH 9 and disintegrates partially to catechol via 1-hydroxy-6-oxo-2-cy- clohexene carboxylic acid anion and 2-hydroxy-3-cyclohexenone irrespective of β-glucosidase activity [4]. Consequently, we ana- lyzed the stability of salicortin under the used assay conditions (incubator) storing it in cell culture medium. During 48 h, sam- ples were taken, and the concentrations of salicortin and catechol were determined by HPLC. 25 μM salicortin decreased to 3.1 μM within 24h. Twelve hours later no salicortin could be detected. Within 8 h the concentration of catechol increased to 8.2 μM. In the following 40 h, the catechol concentrations were relatively stable and varied between 8.2 and 10.9 μM (l " Fig. 3). These re- sults complement the findings of Ruuhola et al. [4] and demon- strate the degradation of salicortin to catechol in the used cell culture medium under assay conditions. Phenolic antioxidants like catechol are described to be inhibitors of proinflammatory pathways. Zheng et al. demonstrated that catechol (2 μg/mL or 18.2 μM, respectively) inhibits the transloca- tion of p65 into the nucleus and additionally the phosphorylation of p38 MAPK in BV-2 microglia cells, resulting in the inhibition of LPS-induced NO-production (IC 50 = 5.56 μM) [5]. Ma and Kinneer showed that antioxidants like catechol and 1,4-dihydroquinone (HQ) are able to decrease the LPS-induced TNF-α production in macrophages (IC 50 [HQ] = 15 μM) and that HQ (100 μM) blocks the LPS induced binding of NF-κB to DNA as indicated by EMSA. It was deduced that antioxidants can activate an Nrf2-like factor Fig. 1 Structural for- mulae of catechol and salicortin. * Dedicated to Prof. Dr. Dr. h. c. Adolf Nahrstedt on the occasion of his 70th birthday 1024 Knuth S et al. Catechol, a Bioactive … Planta Med 2011; 77: 1024–1026 Letters
Transcript of Catechol, a Bioactive Degradation Product of Salicortin, Reduces TNF- α ...
Fig. 1 Structural for-mulae of catechol andsalicortin.
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Catechol, a Bioactive DegradationProduct of Salicortin, Reduces TNF-αInduced ICAM-1 Expressionin Human Endothelial CellsSusanne Knuth1, Helmut Schübel2, Martin Hellemann2,Guido Jürgenliemk1
1 Department of Pharmaceutical Biology, Institute of Pharmacy,University of Regensburg, Regensburg, Germany
2 Hermes Arzneimittel GmbH, Großhesselohe/Munich, Germany
Abstract!
The phenolic glucoside salicortin was isolated from a Willowbark extract, and its ability to reduce the TNF-α induced ICAM-1expression (10 ng/mL, 30min pretreatment with salicortin) wastested in vitro on human microvascular endothelial cells (HMEC-1). After 24 h, 25 µM salicortin decreased the TNF-α inducedICAM-1 expression to 65.9% compared to cells which were treat-ed only with TNF-α. In parallel, the stability of 25 µM salicortinunder assay conditions was determined by HPLC. Within 24 h,the salicortin concentration decreased to 3.1 µM whereas cate-chol, a known NF-κB inhibitor, rose as a metabolite. After 8 h thecatechol concentration was relatively constant and varied be-tween 8.2 and 10.9 µM. Considering this degradation in the in vi-tro test system, 10 µM catechol was added 8 h after TNF-α stimu-lation, and 16 h later the ICAM-1 expression was determined. Inthis setting, the ICAM-1 expression was reduced to 74.8%. This iscomparable to the effect obtained from 25 µM salicortin and indi-cates that its activity is related to the generation of catechol, assalicin, saligenin, and salicylic acid are only marginally active orinactive in this test system in a concentration up to 50 µM. Theseresults indicate catechol as an important bioactive metabolitefrom salicortin.
Key wordsSalix · Salicaceae · salicortin · catechol
Abbreviations!
TNF-α: tumor necrosis factor αICAM-1: intercellular adhesion molecule 1NF-κB: nuclear factor-κBHMEC-1: human microvascular endothelial cells
Supporting information available online athttp://www.thieme-connect.de/ejournals/toc/plantamedica
Herbal remedies containing willow bark extract (Salicis cortex,Salix spec., Salicaceae) are used since ancient times to treat fever,pain, and inflammation [1]. Although salicylic acid, as the in vivometabolite of salicylic alcohol derivatives, was described as theactive principle of willow bark, several studies pointed out thatother ingredients like polyphenols contribute to the overall effectof the drug due to the low achievable salicylic acid plasma con-
* Dedicated to Prof. Dr. Dr. h. c. Adolf Nahrstedt on the occasion of his 70thbirthday
Knuth S et al. Catechol, a Bioactive… Planta Med 2011; 77: 1024–1026
centration [2,3]. To find out if the salicylic alcohol derivativesthemselves could contribute to the anti-inflammatory activityvia hitherto not investigated mechanisms, salicortin was isolatedfrom awillow bark extract, and its ability to reduce the TNF-α in-duced ICAM-1 expression in endothelial cells was investigated.The stability of salicortin in cell culture mediumwas determinedby HPLC.Salicortin was isolated from a willow bark extract by columnchromatography with Sephadex-LH20®, RP-18, and silica gel(l" Fig. 1). In an in vitro assay with human microvascular endo-thelial cells (HMEC-1), 10 to 75 µM salicortin reduced the TNF-αinduced ICAM-1 expression dose-dependently after 24 h of incu-bation (30min pretreatment with test substances; l" Fig. 2). Inthese concentrations no influence on the cell viability was ob-served as indicated by MTT assays. Interestingly, 50 µM of salicin,salicylic acid, and saligenin showed no or only slight effects(l" Fig. 2), whereas the same concentration of salicortin reducedthe ICAM-1 expression to 52.4%.Ruuhola et al. analyzed the in vitro degradation of salicylatesunder different pH values and determined the influence of differ-ent enzymes. Results revealed that salicortin is unstable at pH 9and disintegrates partially to catechol via 1-hydroxy-6-oxo-2-cy-clohexene carboxylic acid anion and 2-hydroxy-3-cyclohexenoneirrespective of β-glucosidase activity [4]. Consequently, we ana-lyzed the stability of salicortin under the used assay conditions(incubator) storing it in cell culture medium. During 48 h, sam-ples were taken, and the concentrations of salicortin and catecholwere determined by HPLC. 25 µM salicortin decreased to 3.1 µMwithin 24 h. Twelve hours later no salicortin could be detected.Within 8 h the concentration of catechol increased to 8.2 µM. Inthe following 40 h, the catechol concentrations were relativelystable and varied between 8.2 and 10.9 µM (l" Fig. 3). These re-sults complement the findings of Ruuhola et al. [4] and demon-strate the degradation of salicortin to catechol in the used cellculture medium under assay conditions.Phenolic antioxidants like catechol are described to be inhibitorsof proinflammatory pathways. Zheng et al. demonstrated thatcatechol (2 µg/mL or 18.2 µM, respectively) inhibits the transloca-tion of p65 into the nucleus and additionally the phosphorylationof p38 MAPK in BV-2 microglia cells, resulting in the inhibition ofLPS-induced NO-production (IC50 = 5.56 µM) [5]. Ma and Kinneershowed that antioxidants like catechol and 1,4-dihydroquinone(HQ) are able to decrease the LPS-induced TNF-α production inmacrophages (IC50 [HQ] = 15 µM) and that HQ (100 µM) blocksthe LPS induced binding of NF-κB to DNA as indicated by EMSA.It was deduced that antioxidants can activate an Nrf2-like factor
Fig. 2 Inhibition of TNF-α induced ICAM-1 expres-sion. Cells were pretreated with different concen-trations of salicortin, 50 µM of saligenin, salicylic ac-id, and salicin (5 µM parthenolide as positive con-trol) for 30min followed by treatment with 10 ng/mL TNF-α for 24 h; HMEC #2–10; n = 3 in duplicates;mean ± SD; *** p < 0.001 vs. neg. control with TNF-α; * p < 0.05 vs. neg. control with TNF-α.
Fig. 3 Degradation of 25 µM salicortin within 48 h incubation with me-dium (incubator 37°C, 5% CO2); n = 3 for eachmeasuring point; mean ± SD.
Fig. 4 Inhibition of TNF-α induced ICAM-1 expression. Cells were pre-treated with 10 ng/mL TNF-α for 8 h followed by incubation with 10 µMcatechol for further 16 h (5 µM parthenolide as positive control).HMEC #2–18; n = 3 in duplicates; mean ± SD; *** p < 0.001 vs. negativecontrol with TNF-α.
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which is able to affect the NF-κB signalling [6]. The link betweenKeap1/Nrf2 and inflammatory pathways and thus the anti-in-flammatory as well as chemopreventive activities of plant anti-oxidants is the subject of several recent publications [7,8]. AsNF-κB strongly affects cell adhesion molecules [9], we looked fora further hint if the observed ICAM-1 expression inhibition iscaused by salicortin itself or its degradation product, catechol.Therefore, 10 µM catechol were added to the endothelial cells8 h after treatment with TNF-α (not 30min before, as usual fortest compounds) according to the concentrations which were ob-served in the degradation study. Sixteen hours later the ICAM-1expression was reduced to 74.8% (l" Fig. 4) which is in the rangeof the value obtained with 25 µM salicortin. Due to the fact thatcatechol raises dynamically in the first 8 h under cell culture con-ditions, this concentration can fully explain the activity of salicor-tin in this in vitro test system.To summarize, salicortin is not stable under the usual cell cultureconditions. Furthermore, these results indicate that not salicortinitself but its degradation product, catechol, is responsible for theability to reduce the TNF-α induced ICAM-1 expression in vitro.
This work points out the importance to check out the stability oftest substances under in vitro assay conditions to exclude wronginterpretation of the obtained data.Further studies will focus on the stability and metabolization ofsalicylic alcohol derivatives in vivo and the influence of metabo-lites beside salicylic acid on the anti-inflammatory activity ofSalicis cortex.
Materials and Methods!
Analytical HPLCHitachi DAD L2455, autosampler L2200, pump L2130, columnoven L2350, software EZChrom Elite; column Hilbar®RT 250-4cartridge with Purospher®Star RP-18e (5 µm). Injection volume:20 µL; mobile phase: 1% THF in water (A), methanol (B); 0–45min: 20% B→ 80% B, 45–50min: 80% B, 50–51min: 80% B→
20% B, 51–60min: 20% B; flow: 0.6mL/min; detection: 270 nm(salicortin), 275 nm (catechol). For calibration, the areas of 1,2.5, 5, 10, 20, 40, 60, and 100 µM salicortin (purity: 96.4%, HPLC)and catechol (Sigma-Aldrich, purity ≥ 99%) in methanol were de-
Knuth S et al. Catechol, a Bioactive… Planta Med 2011; 77: 1024–1026
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termined 3 times. Calibration functions: catechol: y = 20074 ×– 20086; R2 = 0.9936; salicortin: y = 8186.3 × – 29412; R2 =0.9911.
Cell cultureConfluent grown human microvascular endothelial cells (HMEC-1, [10]) were pretreated either with salicylic acid (Sigma-Aldrich,purity ≥ 99%), salicin (Roth, purity ≥ 98%), saligenin (Sigma-Al-drich, purity = 99%), salicortin (isolated), parthenolide (Calbio-chem, purity ≥ 97%, 5 µM, positive control), or medium (ECGM,endothelial cell growth medium (Provitro) + 10% FKS, + antibio-tics, + supplements) as a negative control in 24-well plates. Thirtyminutes later, 10 ng/mL TNF-α (Sigma-Aldrich) were added tostimulate the ICAM-1-expression. After 24 hours of incubation(New Brunswick Scientific, 37°C, 5% CO2), cells were washedwith PBS, removed from the plate with trypsin/EDTA and fixedwith formalin. After incubating with a FITC-labelled mouse anti-body against ICAM-1 (Biozol) for 20min, the fluorescence inten-sity was measured by FACS analysis (Becton Dickinson Facscali-bur™). ICAM-1-expression of cells treated with TNF-α only wasset as 100%. Catechol was added 8 hours after TNF-α incubation,and 16 h later, cells were treated as described above. The influ-ence of catechol and salicortin on the viability of HMEC-1 cellswas determined after 24 h of incubation using an MTT assay ac-cording to Mosman [11] (modified, n = 3 in sextuplicates). Thedetermined IC50 value of catechol on HMEC-1 (IC50 = 329 µM, cal-culated by GraphPad Prism® 4) excluded cytotoxic effects of theemployed 10 µM solution and is in accordance with previous re-sults which showed lacking cytotoxic effects for catechol at a con-centration of 100 µM on macrophages as indicated by the LDH-assay [6]. In case of salicortin, an IC50 value was not determinablefor a rational concentration as viability persisted around 70% us-ing 500 µM salicortin.
Degradation of salicortin500 µL samples of 50 µM salicortin in ECGM per well were kept inthe incubator for 48 hours. After 2, 4, 8, 12, 18, 24, 36, and 48hours, 400 µL were added to 400 µL methanol and centrifuged(centrifuge BR4i multifunction, Jouan industries SAS; 14000U/min, 20min, 10°C) before the supernatant was filtered withNanosep-filters (30 kDa, 14000 U/min, 30min, 10°C) and thesamples were quantified by HPLC.
Statistical analysisEach experiment was performed 3 times. Results are expressedas mean ± SD. Statistical significance between groups was deter-mined by independent samples t-test. A p value < 0.05 was con-sidered as statistically significant.
Supporting informationThe isolation of salicortin is available as Supporting Information.
Knuth S et al. Catechol, a Bioactive… Planta Med 2011; 77: 1024–1026
Acknowledgements!
Special thanks are due to Hermes Arzneimittel for financial sup-port, to Dr. E. Ades, Mr. F. J. Candal of CDC (USA), and to Dr. T. Law-ley of Emory University (USA) for providing the HMEC-1 as wellas to Prof. Dr. Angelika Vollmar for helpful discussions. Prof. Dr.Jörg Heilmann is gratefully acknowledged for fruitful discussions,financial support, and proofreading of the manuscript.
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received August 16, 2010revised January 4, 2011accepted January 11, 2011
BibliographyDOI http://dx.doi.org/10.1055/s-0030-1270722Published online February 8, 2011Planta Med 2011; 77: 1024–1026© Georg Thieme Verlag KG Stuttgart · New York ·ISSN 0032‑0943
CorrespondenceDr. Guido JürgenliemkDepartment of Pharmaceutical BiologyInstitute of PharmacyUniversity of RegensburgUniversitätsstr. 3193053 RegensburgGermanyPhone: + 499419434758Fax: + [email protected]
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