Plant polyphenols as inhibitors of NF-?B induced cytokine production : a potential anti-inflammatory treatment for Alzheimer's disease?

Abstract

Objectives: Neuro-inflammation in Alzheimer's disease occurs as a result of microglial activation that initiates an inflammatory signaling cascade leading to transcription factor NF-?B activation. This results in inflammatory gene expression and elevation of inflammatory mediators. Most plant polyphenolic compounds exert their anti-inflammatory activity by inhibiting proteins in the inflammatory signaling cascade. (E)-Cinnamaldehyde, curcumin, apigenin and (E)- resveratrol are such known plant polyphenolic compounds that exhibit anti-inflammatory activity. However the IC50 values extrapolated from the in vitro data determined in cell cultures should be pharmacologically relevant. The aims of this study were: 1. To identify the effect of (E)-cinnamaldehyde, curcumin, apigenin and (E)-resveratrol to the inflammatory cascade using LPS and IFN-? activated ELAM9 cell line. 2. To identify the effect of the pre incubation time (1hr, 3hrs, 6 hrs, & 9 hrs) on inhibition of NO and TNF a by (E)-cinnamaldehyde, curcumin, apigenin and (E) - resveratrol in LPS and IFN-? activated RAW264.7 and J774.2 macrophage cell lines; 3. To elucidate the structure of the anti-inflammatory compounds in Melicope rubra fruit extract using modern spectroscopic instruments as Melicope rubra fruit extract showed anti-inflammatory activity from preliminary screens. Methods: 1. ELAM9 cell line was treated with (E)-cinnamaldehyde, curcumin, apigenin and (E) - resveratrol and was activated using 50 ng/ml of LPS and 50 U/ml of IFN-?. GFP expression was measured using the flow cytometer. Potency of each compound was calculated using the GraphPad prism software. 2. RAW264.7 and J774.2 murine macrophage cells were pre-incubated with (E)- cinnamaldehyde, curcumin, apigenin and (E)-resveratrol for 1, 3, 6 and 9 hours separately and activated using 10 µg/ml of LPS and 10 U/ml of IFN-?. Cell viability, NO inhibition and TNF-a inhibition were measured for each compound for each hour of pre-incubation. Potency of each compound at each pre incubation hour was calculated using the GraphPad prism software. 3. 100 g of Melicope rubra freeze dried fruit was sequentially extracted using DCM, DCM/MeOH (1:1) and MeOH. DCM extract indicted presence of compounds in LCMS analysis and was identified as the extract with anti-inflammatory activity using RAW 264.7 cells. Griess assay and ELISA were used to measure NO and TNF-a inhibition respectively. The DCM extract was fractionated using the Dionex HPLC and 7 peaks and 2 groups of peaks were collected as fractions and analysed using LCMS and anti-inflammatory assays. The compound in fraction 1 was identified using 1D and 2D NMR spectroscopy. Results: 1. IC50 values ± SD for GFP inhibition by (E)-resveratrol, (E)-cinnamaldehyde, apigenin and curcumin were 164 ± 1.2 µM, 216.9 ± 13.4 µM, 32.5 ± 0.9 µM and 32.4 ± 0.8 µM respectively. 2. Anti-inflammatory potency of apigenin and curcumin did not change with increasing pre incubation time while (E)-cinnamaldehyde showed a decrease in potency and (E)-resveratrol showed an increase in potency when pre-incubated for longer hours before activation. 3. The anti-inflammatory compound isolated from fraction 1 of Melicope rubra fruit DCM extract was identified as 3,7,3'-trimethoxy-5,4'-dihydroxyflavone or pachypodol using NMR, HRMS and LCMS data. The maximum concentration used from fraction 1 to test cytotoxicity and anti-inflammatory effect was 131µM and was found not to be cytotoxic to RAW 264.7 cells. The IC50 values ± SD for NO and TNF-a inhibition by pachypodol was found to be 7.6 ± 0.4 µM and 3.8 ± 0.2 µM respectively. Conclusion: 1. (E)-resveratrol, (E)-cinnamaldehyde, apigenin and curcumin exert their antiinflammatory effect by inhibiting upstream proteins involved in the inflammatory cascade and inhibit transcription of pro inflammatory genes. 2. Most polyphenolic compounds show their maximum potency with 1 hour preincubation in cell based anti-inflammatory assays. However some compounds may show an increased potency when pre-incubated for longer hours. 3. Melicope rubra fruit contains pachypodol which showed a moderate antiinflammatory activity. Its structural features can be used to generate compounds with enhanced anti-inflammatory activity. This study reports TNF-a inhibition by pachypodol for the first time.

Date of Award	2015
Original language	English