Anti-arthritic activity of a lipophilic woad (Isatis tinctoria) extract

• Abstract
• Introduction
• Material and Methods
• Reagents and Chemicals
• Plant material, preparation of extracts and test solutions
• Animals
• Adjuvant arthritis 4, 14
• Clinical assessment, histological radiographic studies of arthritis 5
• Immunohistochemical localization of COX 15
• Determination of TNF-α and IL-1β production in RAW 264.7 macrophages
• Statistics
• Results and Discussion
• Acknowledgement
• References

Abstract

A dichloromethane extract of Isatis tinctoria was tested in the adjuvant-induced arthritis model in rats. The extract (150 mg/kg p. o.) leads to a significant reduction of paw oedema. Radiographic, histological and clinical assessment confirmed reduced damage of cartilage and signs of inflammatory response in comparison to untreated control. No significant differences were observed in the tissular levels of cyclooxygenases1 and -2, and of inducible nitric oxide synthase in Isatis treated and untreated animals. High dose treatment with Isatis extract for two weeks did not result in macroscopic lesions of the gastric mucosa.

Introduction

Rheumatoid arthritis is an inflammatory autoimmune disease which affects approx. 1 % of the population. It is characterized by chronic inflammation of the joints and severe cartilage and bone damage. The etiology is multifactorial, whereby genetic predisposition, environmental and hormonal effects are implicated. The disease is probably triggered by unknown antigens of infectious origin which are presented to immune cells in the joint tissues. Activation of B- and T-cells and release of interleukins (ILs) and tumor nectrosis factor α (TNF-α) ensues, leading to inflammation, formation of rheuma factors and ultimately to irreversible destruction of cartilage and bone tissue. Current pharmacotherapy includes disease modifying antirheumatic drugs (DMARDs), non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and biologicals such as TNF-α antibodies. The treatment focuses on the reduction of pain, inflammation and joint damage. However, the side effects of most drugs may seriously affect the quality of life of patients [1], [2], [3].

Adjuvant-induced arthritis in rats is an experimental disorder first described in 1956 by Pearsons for the in vivo study of new antiarthritic drugs. It is still today the best animal model because it reproduces in many aspects the pathophysiology and biochemistry of the human disease [4]. Adjuvant-induced arthritis is characterized by the apparition of swelling, warmth, erythema, and tenderness in distal joints and tendons of affected animals. Maximal severity is reached between days 16 and 21 after injection of adjuvant. The joint damage associated with adjuvant arthritis is similar to the pathology in patients with rheumatoid arthritis. There is induction of enzymes such as cyclooxygenase-2 (COX-2), and cytokines such as IL-1β and TNF-α in the inflamed joints. Moreover, these mediators trigger the expression of inducible nitric oxide synthase (iNOS) and the subsequent liberation of NO which has a relevant role in the pathogenesis of arthritis [5].

Isatis tinctoria L. (Brassicaceae) is an old European dye plant and medicinal herb which has been used in the context of inflammatory ailments. We, recently, confirmed a promising anti-inflammatory profile of lipophilic leaf extracts in a broad in vitro screening against 20 clinically relevant targets [6]. Constituents responsible for the COX-2, 5-LOX, iNOs, and histamine release inhibitory properties of Isatis extracts have been identified by HPLC-based activity profiling [7], [8], [9], [10], [11]. Among these, tryptanthrin strongly inhibited COX-2 and 5-LOX catalyzed eicosanoid synthesis in various cellular models and in purified enzymes [8] and NO production catalyzed by iNOS [9]. Lipophilic Isatis extracts showed anti-inflammatory and anti-allergic properties in different experimental protocols of acute and subchronic models of inflammation including delayed type hypersensitivity reactions [12]. The same extracts prevented experimentally induced irritant contact dermatitis in healthy volunteers when applied in the eliciting phase [13].

Given the pharmacological profile in vitro, and the in vivo activity in acute and semi-acute inflammation, an evaluation of Isatis extracts in models of chronic inflammation seemed of interest. We here report on the anti-arthritic activity of an extract on Mycobacterium butyricum induced adjuvant arthritis in Lewis rats and its effects on cartilage and bone damage in comparison to a standard NSAID.

Material and Methods

Reagents and Chemicals

Chemical and biochemical reagents were from Fluka Chemika-Biochemika (Buchs, Switzerland), from Baker (Deventer, Holland), from Amersham Biosciences (Madrid, Spain), from Panreac (Barcelona, Spain), and from Sigma Chemical Co. (St. Louis, MO, USA). Primary antibodies were from Santa Cruz (Santa, Cruz, CA, USA).

Plant material, preparation of extracts and test solutions

The same dichloromethane extract as in previous publications was used [12]. The plant material was harvested in 2001 from first year plants (rosette stage) of a defined Isatis tinctoria L. culture, ”Thüringer Waid”, grown on experimental plots of the Agricultural Research Station of Thuringia (TLL), Dornburg, Germany. For further details on the composition of the extract, refer to [12].

Animals

Groups of 14 weeks old female Lewis rats weighing 250 - 300 g were used. Rats were fed a standard diet ad libitum. Housing conditions and all in vivo experiments were approved by the institutional Ethical Committee of the Faculty of Pharmacy according to the guidelines established by the European Union on animal Care (CEE Council 86/609).

Adjuvant arthritis [4], [14]

Adjuvant arthritis was elicited in female Lewis rats by injection of Mycobacterium butyricum (0.1 ml, 10 mg/ml) in mineral oil into the base of the tail. Paw volumes were measured at the beginning of the experiment using a plethysmometer (Ugo Basile; Comerio, Italy). The inflammatory response was evaluated by measuring the volume of both paws at day 17. Animals with paw volumes 1.1 ml larger than normal paws were then randomized into treatment groups. DCM extract (150, 200 and 250 mg/kg), the reference drug indomethacin (1 mg/kg) and vehicle (control) were administered orally once daily. Paw oedemas were measured on days 17 to 23. In parallel, the rats were weighted each day and weight change compared to the control group. Animals were sacrificed by decapitation and arthritis paws amputated above the ankle and placed in 4 % formalin.

Clinical assessment, histological radiographic studies of arthritis [5]

Clinical severity was also determined by measuring the change in the paw volume using a plethysmometer (Ugo Basile). Inflammation was expressed as the increase in paw volume due to arthritis, and the percentage of inhibition was expressed as the reduction in volume with respect to the control group. For microscopic evaluation of histology, the removed paws and knees were fixed in 10 % formalin. The paws were then trimmed, placed in decalcifying solution for 24 h, embedded in paraffin, sectioned at 5 μm, stained with trichromic Van Gieson and studied using light microscopy (Leitz Dialux 22; Wetzlar, Germany).

For radiographic studies, the rats were anaesthetized with sodium pentobarbital (45 mg/kg, i. p.) before they were placed in a radiographic box at a distance of 90 cm from the X-ray source. Radiographic analysis of normal and arthritic hind paws was performed with a 40 kW exposition for 0.01 s (Phillips X12; Munich, Germany). X-ray images were rated according to the following score: 0, no bone damage; score 1, tissue swelling and oedema; score 2, joint erosion; score 3, bone erosion and osteophyte formation.

Immunohistochemical localization of COX [15]

Paraffin sections (4 - 5 μm-thick) were stained following the standard avidin-biotin peroxidase complex technique (LSAB; Dako, Denmark). Antigen retrieval was achieved using EDTA buffer (pH 6) and heating in an autoclave (10 min, 1.5 atm). Primary antibodies included COX-1 (M-20) (Santa Cruz, SC-1754) and COX-2 (M-19) (Santa Cruz, SC-1747). Incubation of primary antibodies was performed at room temperature with a dilution of 1/100 for COX-1 and 1/100 for COX-2. Only cellular cytoplasmic reactivity was considered positive. COX-1 and COX-2 expression was determined by immunohistochemistry. At day 35, the joint’s organs were then trimmed, placed in decalcifying solution for 24 h and 8 μm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3 % H₂O₂ in 60 % methanol for 30 min. The sections were permeabilized with 0.1 % Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2 % normal goat serum in phosphate buffered saline for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 in with avidin and biotin. The sections were then incubated overnight with primary anti-COX-1 (1 : 1000) or with primary anti-COX-2 (1 : 500) with control solutions. Control included buffer alone or non-specific purified rabbit IgG. Specific labelling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex.

Determination of TNF-α and IL-1β production in RAW 264.7 macrophages

Macrophages RAW 264.7 (ECACC; Salisbury, UK) were cultured in DMEM medium containing 2 mM L-glutamine (Gibco; Gaithersburg, MD, USA), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin and 10 % foetal bovine serum (Gibco). Cells were removed from the tissue culture flask using a cell scraper and resuspended until a final relation of 1 x 10⁶ cells ml. Macrophages RAW 264.7 (1 x 10⁶ cells/ml) were co-incubated in 96-well culture plate (200 μl) with 1 μg/ml of LPS at 37 °C for 24 h in the presence of test compound or vehicle. TNF-α and IL-1β was determined in culture supernatant by a specific enzyme immunoassay kit from eBioscience (San Diego, CA, USA).

Statistics

Oedema were expressed as mean ± S.E.M. Inhibition percentages were calculated from the differences between treated and non-treated animals, and compared with the control group treated only with the inflammatory agent. One-way analysis of variance (ANOVA) followed by Dunnett’s t-test for multiple comparisons of unpaired data were used for statistical evaluation. When comparing with control group, values of P less than 0.05 were considered significant. Inhibition percentages (% I) were calculated from the differences between drug treated group and control animal treated only with the inflammatory agent. The inhibitory concentration-50 (IC₅₀) values were calculated from the dose response linear regression plots made with three for IL-1β (25 - 100 μg/ml) and four (TNF-α, 25 - 125 μg/ml) concentrations.

Results and Discussion

In the adjuvant induced arthritis model, the vehicle treated control group developed periarticular erythema, oedema and reduction of paw function. Isatis extract (150 mg/kg) significantly reduced hind paw swelling from day 17 to 23 after challenge by 53 % to 84 %, respectively (Fig. [1] A). Administration of higher doses (200 and 250 mg/kg) did not reduce paw oedema further than the lower dose of 150 mg/kg (data not shown). The reference drug indomethacin (1 mg/kg) led to a reduction in paw volume by 46 % to 80 % between days 17 and 23 (Fig. [1] A). In the initial phase of the arthritis, weight loss was observed in all animals, but the weight of each group stabilized during the later part of the testing. In the extract-treated groups the weight loss was comparable to the control group, and less in the indomethacin group (Fig. [1] B).

Radiographic and histological analysis of the paws was carried out (Fig. [2] and Supporting Information Fig. 1S). Radiography of the tibiotarsal joint of rats revealed a severe inflammation in the control group, with bone resorption and joint erosion (Fig. [2] A₂ ). Radiography of the extract-treated (150 mg/kg) rats showed a decreased joint damage and less soft tissue swelling of the footpad (Fig. [2] A₃ ), similar to the findings for the indomethacin treated group (Fig. [2] A₄ ). The blank group did not present any inflammatory lesion (Fig. [2] A₁ ). The histopathological study of paws from the control group showed the characteristic signs of inflammation and arthritis, with massive mixed cell infiltration, lesions with marked oedema, accesses, cartilage erosion, and bone destruction (Fig. [2] B₂ ). The extract-treated group (150 mg/kg) showed a significant reduction of tissular and articular inflammatory markers (oedema, cells infiltration, articular damage, pannus, cysts, etc.), bone and cartilage erosion (Fig. [2] B₃ . The indomethacin-treated group presented a similar profile than the group treated with the Isatis extract, but with better resolution (Fig. [2] B₄ ), whereas the blank group did not present any inflammatory lesion (Fig. [2] B₁ ). The histological findings are summarized in Table [1].

The immunohistochemical study did not show relevant differences between the treated and non-treated rats. The presence of COX-1 is similar in both the control group and the extract-treated group, whereas some measurable but not significant differences were detected for the inducible enzyme COX-2. Representative tissue preparations stained for COX-1 and COX-2 levels in control and extract treated groups are shown in the Supporting Information, Fig. 2S.

In addition, DCM extract inhibited both TNF-α and IL-1β production in RAW 264.7 macrophages in a concentration dependent manner with an IC₅₀ of 111 and 20 μg/ml, respectively (Fig. [3]).

The lipophilic Isatis shows activity in acute and subchronic in vivo models of inflammation [13], and in adjuvant-induced arthritis in Lewis rats. In the latter model, IL-1β and TNF-α levels are increased in the inflamed joints. These cytokines induce the expression of iNOS and subsequent production of nitric oxide, in addition to a direct noxious effect on cartilage and bone. Nitric oxide, together with prostaglandins (PGs) which are produced as a consequence of an increased expression of COX-2, play crucial roles in the development of induced arthritis. The rats treated with Isatis extract clearly exhibited less joint inflammation and cartilage damage than the untreated control group (Fig. [2] and Supporting Information Fig. 1S). Tissular expression of COX-2 and iNOS, however, were not significantly altered. These findings appear to disagree with earlier in vitro data for Isatis extracts, where a preferential inhibition of COX-2 (IC₅₀ > 40 μg/ml, and reduced expression of iNOS (IC₅₀ 10 μg/ml) was observed [11]. However, the in vitro pharmacological profile of the extract includes activity against other targets [6]. This may explain the anti-arthritic properties observed in vivo despite the lack of a significant effect on expression of COX-2 and iNOS. The fact that the stomach mucosa of extract-treated rats did not show any macromorphological modification or lesion with respect to the control group (data not shown) supports the notion that COX-1 activity does not play a role in the anti-inflammatory activity of Isatis extract in vivo. Moreover, the inhibition of TNF-α and specially IL-1β production by inflammatory cells may be a relevant key for the reduction of the inflammation during the arthritic process.

In conclusion, Isatis tinctoria extract has promising activity in adjuvant-induced arthritis without producing gastric lesions in a high-dose treatment over two weeks. The arthritis model shows an earlier and a later phase [4]. Inhibitors of PG synthesis are effective in both phases, while immunosuppressive agents only act on the latter phase. In an earlier publication, we demonstrated the anti-inflammatory and anti-allergic effect of the same Isatis extract in experimental models of acute inflammation, dermatitis, and in an experimental model of delayed type hypersensitivity (DTH) [12]. In arthritis and DTH, the production of cytokines such as IL-1β and TNF-α is primarily responsible for the tissular damage. In rheumatoid arthritis, NF-κB is overexpressed in the inflamed synovium, leading to increased production of these cytokines [16], [17], [18]. Therefore, the effect of Isatis extract and selected compounds on NF-κB activation and/or IL-1β and TNF-α production in inflamed tissue should be investigated for a further exploration of the potential use of Isatis tinctoria as an anti-inflammatory.

Acknowledgement

Financial support (to JLR) for part of this work by Zeller AG, Romanshorn, Switzerland, is gratefully acknowledged. We are grateful to Dr. A. Vetter and Mrs. A. Biertümpfel, Agricultural Research Station of Thuringia, Dornburg, for the provision of the plant material, and to Dr. B. Weinreich, Adalbert-Raps-Forschungszentrum, Technical University München-Weihenstephan, for preparation of the SFE extract.

References

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Prof. José-Luis Ríos

Department of Pharmacology

Faculty of Pharmacy

University of Valencia

Av. Vicent Andrés Estellés

s/n. 46100 Burjassot

Valencia

Spain

Phone: +34963544973

Fax: +34963544973

Email: riosjl@uv.es

References

• 1 Shmerling R H. Rheumatoid arthritis.  Drugs Today (Barc). 1998;  34 649-53
  CrossrefPubMedSearch in Google Scholar
• 2 Smolen J S, Steiner G. Therapeutic strategies for rheumatoid arthritis.  Nat Rev Drug Discov. 2003;  2 473-88
  CrossrefPubMedSearch in Google Scholar
• 3 Lundberg I E, Grundtman C, Larsson E, Klareskog L. Corticosteroids - from an idea to clinical use.  Best Pract Res Clin Rheumatol. 2004;  18 7-19
  CrossrefPubMedSearch in Google Scholar
• 4 Taurog J D, Argentieri D C, McReynolds R A. Adjuvant arthritis.  Methods Enzymol. 1988;  162 339-55
  PubMedSearch in Google Scholar
• 5 Cuzzocrea S, Mazzon E, Bevilaqua C, Costantino G, Britti D, Mazullo G. et al . Cloricromene, a coumarine derivative, protects against collagen-induced arthritis in Lewis rats.  Br J Pharmacol. 2000;  131 1399-407
  CrossrefPubMedSearch in Google Scholar
• 6 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
• 7 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
• 8 Danz H, Stoyanova S, Thomet O AM, Simon H U, Dannhardt G, Ulbrich H. et al . Inhibitory activity of tryptanthrin on prostaglandin and leukotriene synthesis.  Planta Med. 2002;  68 875-80
  Thieme ConnectPubMedSearch in Google Scholar
• 9 Ishihara T, Kohno K, Ushio S, Ikeda M, Kurimoto M. Tryptanthrin inhibits nitric oxide and prostaglandin E₂ synthesis by murine macrophages.  Eur J Pharmacol. 2000;  407 197-204
  CrossrefPubMedSearch in Google Scholar
• 10 Rüster G -U, Hoffmann B, Hamburger M. Inhibitory activity of indolin-2-one derivatives on compound 48/80-induced histamine release from mast cells.  Pharmazie. 2004;  59 236-7
  PubMedSearch in Google Scholar
• 11 Oberthür C, Jäggi U, Hamburger M. HPLC-based activity profiling of a lipophilic Isatis tinctoria leaf extract for 5-lipoxygenase inhibitory activity.  Fitoterapia. 2005;  76 324-32
  CrossrefPubMedSearch in Google Scholar
• 12 Recio M C, Cerdá-Nicolás M, Potterat O, Hamburger M, Ríos J L. Anti-inflammatory and anti-allergic activity in vivo of lipophilic Isatis tinctoria extracts and tryptanthrin.  Planta Med. 2006;  72 539-46
  Thieme ConnectPubMedSearch in Google Scholar
• 13 Heinemann C, Schliemann-Willers S, Oberthür C, Hamburger M, Elsner P. Prevention of experimentally induced irritant contact dermatitis by extracts of Isatis tinctoria compared to pure tryptanthrin and its impact on UVB-induced erythema.  Planta Med. 2004;  70 385-90
  Thieme ConnectPubMedSearch in Google Scholar
• 14 De León E J, Alcaraz M J, Domínguez J N, Charris J, Terencio M C. 1-(2,3,4-trimethoxyphenyl)-3-(3(2-chloroquinolinyl))-2-propen-1-one, a chalcone derivative with analgesic, anti-inflammatory and immunomodulatory properties.  Inflamm Res. 2003;  52 246-57
  PubMedSearch in Google Scholar
• 15 Llombart-Bosch A, Navarro S. Immunohistochemical detection of EWS and FLI-1 proteins in Ewing sarcoma and primitive neuroectodermal tumors: comparative analysis with CD99 (MIC-2) expression.  Appl Immunohistochem Mol Morphol. 2001;  9 255-60
  CrossrefPubMedSearch in Google Scholar
• 16 Ghosh S, May M J, Kopp E B. NF-κB and rel proteins: Evolutionarily conserved mediators of immune responses.  Annu Rev Immunol. 1998;  16 225-60
  CrossrefPubMedSearch in Google Scholar
• 17 Tak P T, Firestein G S. NF-κB: a key role in inflammatory diseases.  J Clin Invest. 2001;  107 7-11
  CrossrefPubMedSearch in Google Scholar
• 18 Bremner P, Heinrich M. Natural products as targeted modulators of the nucleat factor-κB pathway.  J Pharm Pharmacol. 2002;  54 453-72
  CrossrefPubMedSearch in Google Scholar

Prof. José-Luis Ríos

Department of Pharmacology

Faculty of Pharmacy

University of Valencia

Av. Vicent Andrés Estellés

s/n. 46100 Burjassot

Valencia

Spain

Phone: +34963544973

Fax: +34963544973

Email: riosjl@uv.es

• Supplementary Material