Effects of flavonoids on the fatty acid amide hydrolase activity of rat liver in vitro

Stephen Alexander, Annie Patel, Michael Garle. University of Nottingham, Nottingham, United Kingdom.

Flavonoids are polyphenolic antioxidants, abundant in the human diet. They are thought to provide beneficial effects on a number of bodily properties, such as cardiovascular and cerebrovascular events, as well as longevity. Amongst the molecular targets suggested for flavonoids are the eicosanoid-metabolising enzymes, cyclooxygenase and lipoxygenase. Since the endocannabinoids anandamide and 2-AG are themselves eicosanoid derivatives, we have investigated whether fatty acid amide hydrolase (FAAH) activity can be modified in vitro in the presence of flavonoids.

Rat (male Wistar, 170-300 g) liver microsomes were prepared as previously described (De Bank et al., 2005) and stored as aliquots at -80°C. FAAH activity of Triton X-100 solubilised rat liver preparations was assessed over 30 min in the presence of inhibitors and 100 μM oleamide (as substrate) in a final volume of 100 μL, containing 10 % (v/v) DMSO and 0.05 (v/v) Triton X-100 in 0.2 M phosphate buffer, pH 7.4 (Patel et al., 2007). Data presented are means ± SEM of single replicate determinations using four separate liver preparations and were analysed for statistical significance using 1-way ANOVA and Bonferroni’s multiple comparison.

The flavonoids tested at 100 μM had a rank order of effectiveness (% control; **P<0.01): kaempferol (23 ± 2**) > luteolin (29 ± 1**), 3,7-dihydroxyflavone (32 ± 3**), baicalein (34 ± 1**) > morin (37 ± 2**), rutin (40 ± 6**), epigallocatechin gallate (40 ± 11**), genistein (42 ± 2**), catechin (45 ± 14**), fisetin (51 ± 2**), quercetin (51 ± 2**), 4’-hydroxyflavanone (51 ± 4**), epicatechin (52 ± 1*), chrysin (53 ± 8**), 3,6-dihydroxyflavone (55 ± 5**), baicalin (57 ± 3**), apigenin (57 ± 12**), myricetin (60 ± 3**) > daidzein (72 ± 3**), 7-hydroxyflavanone (74 ± 3**), genistein (74 ± 4**), flavone (78 ± 10), 6-hydroxyflavanone (85 ± 5), flavanone (88 ± 5).

Using a range of concentrations of selected flavonoids allowed calculation of a rank order of potency (pIC50 values): kaempferol (4.6 ± 0.2) > morin (4.4 ± 0.1) > luteolin (4.3 ± 0.2) > 3,7-dihydroxyflavone (4.2 ± 0.1) > baicalein (4.1 ± 0.2), quercetin (4.1 ± 0.1).

Whilst the parent, non-hydroxylated flavonoids, flavone and flavanone, were inactive, the polyhydroxylated derivatives showed a range of activities. Glycosylation in the 3- position (quercetin vs. rutin and epigallocatechin vs. catechin/epicatechin) appeared to have less of an effect than in the 7- position (baicalein vs. baicalin), whilst 7-hydroxylation appeared to enhance activity, although being insufficient alone to confer high potency. Thus, the flavonoids represent a novel family of non-cannabinoid plant-derived products with activity on the cannabinoid system.

De Bank PA, et al. (2005) Biochem. Pharmacol. 69, 1187-93.

Patel A, et al. (2007) Proceedings of the BPS Oxford Meeting

We thank the MRC for a studentship (AP).