Butrin, isobutrin, and butein from medicinal plant Butea monosperma selectively inhibit nuclear factor-kappaB in activated human mast cells: suppression of tumor necrosis factor-alpha, interleukin (IL)-6, and IL-8

Abstract

Activation of mast cells in rheumatoid synovial tissue has often been associated with tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8 production and disease pathogenesis by adjacent cell types. Butea monosperma (BM) is a well known medicinal plant in India and the tropics. The aim of this study was to examine whether a standardized extract of BM flower (BME) could inhibit inflammatory reactions in human mast cells (HMC) using activated HMC-1 cells as a model. Four previously characterized polyphenols--butrin, isobutrin, isocoreopsin, and butein--were isolated from BME by preparative thin layer chromatography, and their purity and molecular weights were determined by liquid chromatography/mass spectrometry analysis. Our results showed that butrin, isobutrin, and butein significantly reduced the phorbol 12-myristate 13-acetate and calcium ionophore A23187-induced inflammatory gene expression and production of TNF-alpha, IL-6, and IL-8 in HMC-1 cells by inhibiting the activation of NF-kappaB. In addition, isobutrin was most potent in suppressing the NF-kappaB p65 activation by inhibiting IkappaBalpha degradation, whereas butrin and butein were relatively less effective. In vitro kinase activity assay revealed that isobutrin was a potent inhibitor of IkappaB kinase complex activity. This is the first report identifying the molecular basis of the reported anti-inflammatory effects of BME and its constituents butrin, isobutrin, and butein. The novel pharmacological actions of these polyphenolic compounds indicate potential therapeutic value for the treatment of inflammatory and other diseases in which activated mast cells play a role.

Fig. 1.

Screening of BM polyphenols. HPLC chromatogram of the whole BME (A) and the preparative TLC-purified fractions of BME (B–E). Inset, mass spectrometry-chromatogram of TLC-purified fractions of BME and chemical structures of BM polyphenols used in this study. B, butrin: molecular weight 596, molecular formula C₂₇H₃₂O₁₅, and International Union of Pure and Applied Chemistry (IUPAC) name (2S)-2-[4-hydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]-7-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-hydroxymethyl)oxan-2-yl]oxy-2,3-dihydrochromen-4-one. C, isobutrin: molecular weight 596, molecular formula C₂₇H₃₂O₁₅, IUPAC name (E)-1-[2-hydroxy-4-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]-3-[4-hydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]prop-2-en-1-one isobutrin. D, isocoreopsin: molecular weight 434, molecular formula C₂₇H₃₂O₁₅, IUPAC name (2S)-2-(3,4-dihydroxyphenyl)-7-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]ox y-2,3-dihydrochromen-4-one. E, butein: molecular weight 272, molecular formula C₁₅H₁₂O₅, IUPAC name (E)-1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)prop-2-en-1-one.

Fig. 2.

Antioxidant potential of BM polyphenols. A, DPPH radical-scavenging capacity of BM polyphenols. The radical scavenging ability of BM polyphenols was analyzed by measuring their inhibitory effects on the absorbance of DPPH radicals at 517 nm. Ascorbic acid was used as a positive control. The reaction was performed in triplicates, and results were expressed as percentage of inhibition of the absorbance of the DPPH radical ± S.E.M. B, ORAC of BM polyphenols. The antioxidant activity of BM polyphenols by ORAC was conducted in microplates using fluorescein as the fluorescent probe. Ascorbic acid was used as positive control. The reaction was performed in triplicates and results were expressed as micromoles of Trolox equivalence per microgram of dried BME and its polyphenols. C, percentage of inhibition of NO radicals in the presence of BM polyphenols. The radical scavenging ability of BM polyphenols was analyzed by measuring their inhibitory effects on the absorbance of the NO reaction product at 540 nm. Ascorbic acid was used as a positive control. The reaction was performed in triplicates and results were expressed as percentage of inhibition of the absorbance of NO reaction product ± S.E.M. Bars with the same letter do not differ statistically (p < 0.05).

Fig. 3.

Effect of BM polyphenols and specific inhibitor of NF-κB on TNF-α, IL-6, and IL-8 gene expression and production in PMACI-stimulated human mast cells. HMC-1 cells were pretreated with butrin, isobutrin, isocoreopsin, butein, or BME (1–10 μg/ml) for 2 h and stimulated by PMA (40 nM) plus A23187 (1 μM) for 8 h (A, C, and E) or 24 h (B, D, and F). Gene expression of TNF-α (A), IL-6 (C), and IL-8 (E) was determined by quantitative RT-PCR normalized to GAPDH and then compared with the levels present in untreated cells. Level of TNF-α (B), IL-6 (D), and IL-8 (F) in the culture medium was quantified by sandwich ELISA. Concentration of NF-κB inhibitor (parthenolide) used in these studies was 50 μM. Results are representative (mean ± S.E.M.) of four independent experiments and differ without a common letter (p < 0.05).

Fig. 4.

Effect of BM polyphenols on the activation of NF-κB in PMACI-stimulated HMC-1 cells. Cells were pretreated with butrin, isobutrin, isocoreopsin, butein, or BME (1–10 μg/ml) for 2 h before PMA (40 nM) plus A23187 (1 μM) stimulation. A, NF-κB p65 was determined in nuclear extracts by highly sensitive and specific ELISA (Assay Designs). The potent inhibitor for IκB kinase, parthenolide (50 μM), was used as a positive control. TNF-α-treated HeLa cell extract (supplied with kit) was also used as a positive control. The assay is developed with a chemiluminescent substrate and the signal is detected using a luminometer (Lumat LB 9507; Berthold Technologies). Nuclear NF-κB p65 activity was expressed as relative light units (RLU). Results are representative (mean ± S.E.M.) of four independent experiments and differ without a common letter (p < 0.05). B, IκBα degradation was analyzed by Western immunoblotting using antibodies specific for the IκBα (Cell Signaling Technology Inc.). β-Actin was used as protein loading control. C, band images were digitally captured and the band intensities (pixels/band) were obtained using the Un-Scan-It software and are expressed in average pixels. Data shown is cumulative of three experiments and the optical density values are mean ± S.D. and differ without a common letter (p < 0.05). D, BM polyphenols inhibited the IKKβ kinase activity in vitro. IKKβ kinase activity was determined in the absence or presence of BM polyphenols (0–1000 ng/ml) by using the HTScan IKKβ kinase assay kit (Cell Signaling Technology Inc.). Each bar represents the mean ± S.E.M. of three independent experiments.